KR102774944B1 - Low molecular weight silk compositions and stabilizing silk compositions - Google Patents

Abstract

The present disclosure provides certain silk-fibroin compositions having particular features and/or characteristics. In some embodiments, the present disclosure provides low molecular weight compositions. In some embodiments, the present disclosure provides silk fibroin compositions comprising an active (e.g., biological) agent or component. In some embodiments, the present disclosure provides low molecular weight silk fibroin compositions comprising an active (e.g., biological) agent or component. In some embodiments, the active agent is stabilized in the silk composition, for example, for a period of time and/or under certain conditions or circumstances. In some embodiments, the components present in the silk fibroin composition can be analyzed and/or characterized. In some embodiments, the components present in the silk fibroin composition can be recovered from the composition.

Description

{LOW MOLECULAR WEIGHT SILK COMPOSITIONS AND STABILIZING SILK COMPOSITIONS}

Government support

This invention was made with government support under grant SB112-005 awarded by DARPA and grant P41 EB002520 awarded by NIH. The government has certain rights in the invention.

Background Technology

Stabilization and subsequent recovery of active agents and/or biological samples is an important feature in many applications, since active agents and/or biological samples are generally sensitive and unstable to changes in ambient conditions, such as temperature, humidity and/or light. Even if an active agent or biological sample is found to be useful for a given reaction, its application is often hampered by a lack of long-term stability under the manufacturing conditions.

Various methods have been utilized or studied to stabilize active agents (e.g., therapeutic proteins including enzymes, vaccines) and/or biological samples (e.g., blood and/or blood components), including cold-chain storage, lyophilization, covalent immobilization, and cellulose-based technologies. However, all of these methods have drawbacks such as intense energy requirements or poor recovery of the active agent and/or biological sample. These drawbacks further limit their options for on-demand or point-of-care applications. Accordingly, there is a need for improved techniques and/or products or novel materials that can stabilize active agents and/or biological samples (e.g., at ambient conditions) and enable recovery of various analytes from the stabilized sample in sufficient quantities for therapeutic, diagnostic, detection and/or analytical purposes.

outline

The present disclosure provides certain silk fibroin compositions having novel and/or unexpected structural and/or functional features and/or properties. The present disclosure provides methods of making and/or using such compositions, as well as articles made of or from them. In some embodiments, the provided compositions provide an active (e.g., biological) agent or ingredient. In some embodiments, the active agent or ingredient is stabilized in the composition as compared to other comparable conditions, such as, for example, the silk fibroin and/or lack of a particular structural and/or functional feature and/or property.

Among other things, the various aspects of the embodiments described herein stem from the discovery that low molecular weight silk fibroin-based silk materials provide unique material properties that make them suitable for many applications. As described in detail below, the low molecular weight silk fibroin compositions described herein provide certain material characteristics that are distinct or improved when compared to conventional silk fibroin-based materials. Thus, in one aspect, provided herein is a low molecular weight silk fibroin composition comprising a population of silk fibroin fragments having varying molecular weights, the population of silk fibroin fragments being characterized in that no more than 15% of the total number of silk fibroin fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total number of silk fibroin fragments in the population have a molecular weight within a specified range, wherein the specified range is from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa.

In other words, the low molecular weight silk fibroin compositions described herein can comprise a population of silk fibroin fragments having varying molecular weights, wherein the population of silk fibroin fragments has a molecular weight greater than 200 kDa, wherein no more than 15% of the total moles of the silk fibroin fragments in the population have a molecular weight within a particular range, wherein the particular range is from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa.

The low molecular weight silk fibroin composition can exist in a variety of forms. In some embodiments, the low molecular weight silk fibroin composition is an aqueous solution. The inventors have surprisingly discovered that agents (e.g., but not limited to, nucleic acids such as proteins, DNA, RNA and/or modifications thereof, therapeutic agents, vaccines) or samples (e.g., biological samples) incorporated or mixed with the low molecular weight silk fibroin solution described herein can be stabilized for a period of time under a wide range of temperatures. In some embodiments, the agents or samples incorporated or mixed with the low molecular weight silk fibroin solution can be stabilized for a period of time, for example, for more than 24 hours, under a variety of conditions, including, for example, ambient conditions (e.g., room temperature) and/or elevated temperature conditions (e.g., 60° C.).

In some embodiments, the low molecular weight silk fibroin composition can be in a solid state form, for example, a silk fibroin article. Examples of silk fibroin articles can include, but are not limited to, films, sheets, gels or hydrogels, meshes, mats, non-woven mats, fabrics, scaffolds, tubes, slabs or blocks, fibers, particles, powders, three-dimensional structures, implants, foams or sponges, needles, lyophilized articles, and any combination thereof. In some embodiments, the silk fibroin articles can be bioresorbable implants, tissue scaffolds, sutures, reinforcing materials, medical devices, coatings, building materials, wound dressings, tissue sealants, fabrics, textile products, and any combination thereof.

In some embodiments, the low molecular weight silk fibroin article is readily reconstituted into a low molecular weight silk fibroin solution for further processing. In some embodiments, the low molecular weight silk fibroin article described herein can have a solubility of greater than or equal to 5% in an aqueous solution. That is, greater than or equal to 5% of the total weight or volume of the low molecular weight silk fibroin article can be dissolved or reconstituted in an aqueous solution. In some embodiments, the low molecular weight silk fibroin article described herein can have a solubility of greater than or equal to 5%, including, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more. In some embodiments, the low molecular weight silk fibroin article described herein can be solubilized or reconstituted at room temperature. In some embodiments, the low molecular weight silk fibroin articles described herein can be solubilized or reconstituted in water, such as deionized water, at room temperature. In some embodiments, the low molecular weight silk fibroin articles can be used in on-demand applications.

In some embodiments, the low molecular weight silk fibroin articles described herein can have improved solubility in aqueous solution relative to a reference silk fibroin composition (e.g., a conventional silk-fibroin based material). In some embodiments, the solubility of the low molecular weight silk fibroin article in aqueous solution can be improved by at least about 5% relative to the reference silk fibroin composition. For example, the solubility of the low molecular weight silk fibroin article in aqueous solution can be improved by at least 5% relative to the solubility of the reference silk fibroin composition, including, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more. In some embodiments, the solubility of the low molecular weight silk fibroin article in the aqueous solution can be improved by at least about 1.1 fold compared to the solubility of a reference silk fibroin composition. For example, the solubility of the low molecular weight silk fibroin article in the aqueous solution can be improved by at least about 1.1 fold compared to a reference silk fibroin composition, including, for example, at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold or more. In some embodiments, the aqueous solution can be water. In some embodiments, the aqueous solution can be a buffer solution, such as, but not limited to, a phosphate buffer solution.

In some embodiments, the low molecular weight silk fibroin articles described herein can be used to trap at least one agent or sample (e.g., a biological sample) for stabilization of the agent or sample and subsequent recovery thereof. In some embodiments, a portion or original load of the agent or sample can be recovered from at least a portion or an aliquot of the low molecular weight silk fibroin article by dissolving the portion or aliquot of the low molecular weight silk fibroin article in an aqueous solution. In some embodiments, the portion or aliquot of the low molecular weight silk fibroin article can be dissolved in an aqueous solution (e.g., but not limited to, water, such as deionized water or a buffered solution) at room temperature. In some embodiments, the active agent or sample (e.g., a biological sample) trapped within the low molecular weight silk fibroin article described herein can have a recovery of at least 5% in the aqueous solution. That is, greater than or equal to 5% of the original load of the active agent or sample can be recovered from the low molecular weight silk fibroin article in solution or liquid form. In some embodiments, the active agent or sample (e.g., a biological sample) trapped within the low molecular weight silk fibroin article described herein can have a recovery of greater than or equal to 5% in aqueous solution, including, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more.

In some embodiments, the low molecular weight silk fibroin article can provide improved recovery of an agent or sample trapped therein as compared to the recovery of an agent or sample trapped within a reference silk fibroin composition. In some embodiments, the recovery of an agent or sample trapped within the low molecular weight silk fibroin article can be improved by at least about 5% as compared to the recovery of an agent or sample trapped within a reference silk fibroin composition. For example, the recovery of an agent or sample trapped within the low molecular weight silk fibroin article can be improved by at least 5% as compared to the recovery of an agent or sample trapped within a reference silk fibroin composition, including, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more. In some embodiments, the recovery of an agent or sample trapped within the low molecular weight silk fibroin article can be improved by at least about 1.1-fold compared to the recovery of an agent or sample trapped within a reference silk fibroin composition. For example, the recovery of an agent or sample trapped within the low molecular weight silk fibroin article can be improved by greater than about 1.1-fold, including, for example, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold or more. In some embodiments, the aqueous solution can be a buffered solution, such as, but not limited to, a phosphate buffered solution. In some embodiments, the use of a buffer solution (e.g., but not limited to, a phosphate buffer solution) to reconstitute the low molecular weight silk fibroin articles described herein can increase the recovery of active agents or samples incorporated into the low molecular weight silk fibroin articles when compared to the use of a non-buffered solution (e.g., water).

In various embodiments of the different aspects described herein, the reference silk fibroin composition can be a composition or mixture produced by refining silk cocoons at atmospheric boiling temperature for about 60 minutes or less, for example, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 10 minutes or shorter. In one embodiment, the reference silk fibroin composition can be a composition or mixture produced by refining silk cocoons at atmospheric boiling temperature in an aqueous sodium carbonate solution for about 60 minutes or less, for example, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 10 minutes or shorter.

Another aspect provided herein relates to a storage-stable composition comprising a silk fibroin article, wherein the silk fibroin article comprises one or more embodiments of the low molecular weight silk fibroin compositions described herein. In some embodiments, the storage-stable composition is storage-stable, such that the material properties of the silk fibroin article remain stable for at least one month. In some embodiments, the storage-stable composition can comprise the low molecular weight silk fibroin composition in the form of particles, including a powder.

In general, the present disclosure encompasses the recognition that certain silk fibroin compositions are particularly useful for the incorporation of active (e.g., biological) and/or labile entities. In some embodiments, silk compositions comprising active and/or labile agents, such as biological samples or components thereof, are provided. In some embodiments, provided silk compositions (e.g., comprising low molecular weight silk fibroin compositions) stabilize active and/or labile agents (e.g., stabilize biological samples or components thereof).

While low molecular weight silk fibroin particles, including powders, can be produced by any art recognized method, in some embodiments, the low molecular weight silk fibroin particles can be produced by a process comprising lyophilizing a low molecular weight silk fibroin solution, thereby forming a lyophilized low molecular weight silk fibroin material or particle. In some embodiments, the lyophilized low molecular weight silk fibroin material or particle can be further reduced into smaller particles. In one embodiment, the lyophilized low molecular weight silk fibroin material or particle can be further milled to produce smaller particles or powder. Thus, another aspect provided herein is a storage-stable reagent comprising a low molecular weight silk fibroin powder. In some embodiments, the low molecular weight silk fibroin powder can be a lyophilized powder. The storage-stable reagents may be provided as part of a kit or in a container, for example, a vial, a syringe, or a sample collection container, for example, a blood collection container, or a multi-well plate.

Various embodiments described herein provide storage-stable compositions comprising one or more embodiments of a low molecular weight silk fibroin composition described herein and an agent that is desirable to be stabilized, wherein the agent is incorporated or mixed into the low molecular weight silk fibroin composition. In some embodiments, the agent that is desirable to be stabilized can be an active agent. For example, in some embodiments, a protein, a peptide, a nucleic acid molecule, and/or a modified nucleic acid molecule can be stabilized in the low molecular weight silk fibroin composition. In some embodiments, the agent that is desirable to be stabilized can be a therapeutic agent. In some embodiments, the agent that is desirable to be stabilized can be a diagnostic marker for a disease or disorder. In some embodiments, the agent that is desirable to be stabilized can be a sample that is to be analyzed for one or more components present in the sample. In some embodiments, the sample can be a biological sample.

Another aspect provided herein relates to a silk fibroin article comprising one or more embodiments of the low molecular weight silk fibroin composition described herein and a substrate. The low molecular weight silk fibroin composition can be dispersed in the substrate, deposited on a substrate, or a combination thereof.

Another aspect provided herein is a method of forming a silk fibroin solution comprising dissolving one or more embodiments of the low molecular weight silk fibroin compositions described herein in a liquid, wherein the silk fibroin solution is a homogeneous solution. In one embodiment, the liquid is water, such as, but not limited to, deionized water. As used herein, the term "homogeneous solution" generally refers to a solution having a uniform appearance or composition throughout the solution. For example, the size of the silk fibroin fragments or particles in the homogeneous silk fibroin solution is generally too small to be observed or detected, for example, with the naked eye. In other words, the homogeneous silk fibroin solution is substantially free of silk fibroin aggregates (e.g., insoluble silk fibroin fragments, silk fibroin particles, and/or clumps). Examples of silk fibroin aggregates may include, but are not limited to, full-length silk fibroin molecules, larger silk fibroin fragments, silk fibroin particles or clusters formed by the aggregation or coagulation of smaller silk fibroin fragments, and any combination thereof.

Another aspect provided herein relates to a method of stabilizing a formulation for a period of time under certain conditions in one or more embodiments of the silk fibroin composition described herein. In some embodiments, the formulation can be stabilized under ambient conditions.

A further aspect provided herein is a method of recovering at least one active agent comprising: (a) providing one or more embodiments of a low molecular weight silk fibroin composition described herein, one or more embodiments of a storage-stable composition described herein, or one or more embodiments of a storage-stable composition described herein, wherein the at least one active agent is stabilized within the composition; and (b) dissolving at least a portion of the composition in water, thereby forming a sample solution comprising silk fibroin and a detectable or measurable amount of the at least one active agent.

Another aspect provided herein is a method of modulating at least one property of a silk fibroin composition, comprising varying the weight ratio of silk fibroin fragments within the composition having a molecular weight greater than 200 kDa to silk fibroin fragments within the composition having a molecular weight within a particular range, wherein the particular range is from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa.

Another aspect provided herein is a method of controlling the size of silk fibroin particles comprising two steps: (a) varying the weight ratio of silk fibroin fragments in the solution having a molecular weight greater than 200 kDa to silk fibroin fragments in the solution having a molecular weight within a particular range, wherein the particular range is from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa; and (b) forming silk fibroin particles from the silk fibroin solution.

Silk fibroin is an example of a polypeptide having a portion or portions of an amino acid sequence capable of assuming a beta-sheet secondary structure. For example, a silk fibroin structure generally comprises an amino acid sequence, wherein portions or portions of the sequence are characterized by alternating glycine and alanine, or only alanine. Without wishing to be bound by theory, it is believed that this arrangement allows the fibroin molecule to self-assemble into a beta-sheet conformation. Thus, in another aspect, provided herein is a composition comprising a population of polypeptide fragments having portions or portions of an amino acid sequence characterized by alternating glycine and alanine, or only alanine, and having a molecular weight ranging from about 3.5 kDa to about 120 kDa or from about 5 kDa to about 125 kDa. In some embodiments, the composition is characterized in that: no more than 15% of the total number of polypeptide fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total number of polypeptide fragments in the population have a molecular weight within a particular range, wherein the particular range is from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa.

In another aspect, the silk fibroin in the compositions and/or methods described herein can be substituted for or combined with other non-silk polypeptides that comprise a beta sheet structure or have a propensity to form such structures based on their amino acid sequence. Accordingly, also provided herein are polypeptide compositions comprising a population of beta-sheet forming polypeptide fragments. In some embodiments, the population of beta-sheet forming polypeptide fragments described herein can have a variety of molecular weights characterized by: no more than 15% of the total number of beta-sheet forming polypeptide fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total number of beta-sheet forming polypeptide fragments in the population have a molecular weight in a particular range, wherein the particular range is from about 3.5 kDa to about 120 kDa or from about 5 kDa to about 125 kDa.

As used herein, the term "beta-sheet forming polypeptide" refers to a polypeptide having a portion or portions of an amino acid sequence that adopts a beta-sheet secondary structure. In some embodiments, a beta-sheet forming polypeptide can be selected based on its propensity to form such a structure based on its beta sheet structure or amino acid sequence.

In some embodiments, the beta-sheet forming polypeptide can have an amphiphilic nature (i.e., having both hydrophilic and hydrophobic properties and/or moieties). The amphiphilic polypeptide can be obtained from a single source (e.g., a naturally occurring protein) and can comprise both a hydrophobic module or region and a hydrophilic module or region within the polypeptide, such that the single polypeptide itself is inherently amphiphilic. In some embodiments, the hydrophobic module or region and the hydrophilic module or region can be fused or coupled together to form an amphiphilic entity. Such "fusion" or "chimeric" polypeptides can be produced using recombinant techniques, chemical coupling, or both.

In some embodiments, the beta-sheet forming polypeptide can comprise a portion or parts of the amino acid sequence of a polypeptide selected from the following list: fibroin, actin, collagen, catenin, claudin, coilin, elastin, elanin, extensin, fibrillin, lamin, laminin, keratin, tubulin, structural proteins of viruses, zein protein (seed storage protein) and any combination thereof.

In some embodiments, the beta-sheet forming polypeptide can comprise a protein regenerated (e.g., purified) from a natural source, a recombinant protein produced in a heterologous system, a synthetically or chemically produced peptide, or a combination thereof.

In some embodiments, the beta-sheet forming polypeptide can comprise a portion or parts of the amino acid sequence of a polypeptide corresponding to any one of the lists provided above, with or without one or more sequence variations, as compared to a native or wild-type counterpart. For example, in some embodiments, such variations can exhibit an overall sequence identity of at least 85%, for example, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, as compared to a wild-type sequence.

This patent or application file contains at least one drawing made in color. This patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the required fee.
Figure 1 shows a process flow diagram for the solubility study of refined silk for 10, 20, 30, or 60 minutes. Freeze-drying conditions and solubilization ratios are also provided.
Figure 2 shows the method used to calculate the percentage of four molecular weight bands (greater than 200 kDa, 200 to 116.3 kDa, 116.3 to 66.3 kDa, and 66.3 to 36.5 kDa). For this gel, 7.5 μg of protein in solution was run under reducing conditions on a NuPAGE® Novex® 3-8% Tris-acetate gel (Invitrogen) using Mark12™ Unstained Standard (range 31-200 kDa, Invitrogen). Figure 2a shows representative images of SDS-PAGE analysis for silks smelted for 10, 20, 30, 60 minutes, or autoclaved for 60 minutes. Figure 2b shows a representative density image analysis performed on an electrophoresis gel. Raw pixel intensities are plotted against molecular weight. The percentage of each molecular weight band can be calculated by dividing the summed intensity of a particular molecular weight band by the total summed intensity of all molecular weight bands.
Figure 3a is an image of an SDS-PAGE gel for a silk fibroin solution produced from silk foam stored at 4°C. Figure 3b shows the molecular weight distribution of silk fibroin that underwent primary drying from the gel data of Figure 3a. Figure 3c shows the molecular weight distribution of silk fibroin that underwent primary and secondary drying from the gel data of Figure 3a.
Figure 4a is an image of an SDS-PAGE gel for a silk fibroin solution produced from silk foam stored at 22°C. Figure 4b shows the molecular weight distribution of silk fibroin that underwent primary drying from the gel data of Figure 4a. Figure 4c shows the molecular weight distribution of silk fibroin that underwent primary and secondary drying from the gel data of Figure 4a.
Figure 5a is an image of an SDS-PAGE gel for a silk fibroin solution produced from silk foam stored at 37°C. Figure 5b shows the molecular weight distribution of silk fibroin that underwent primary drying from the gel data of Figure 5a. Figure 5c shows the molecular weight distribution of silk fibroin that underwent primary and secondary drying from the gel data of Figure 5a.
Figure 6 shows the silk foam solubility versus loading. Silk fibroin solutions generated from various boiling times (10-60 min boiling, MB) were lyophilized using the as-purified solutions (a, b) and autoclaved solutions (c, d) according to the process flow shown in Figure 1. Either 20, 40, or 80 mg of the milled powder was added to deionized water to make a final volume of 1 mL. After centrifugation at 1000 rpm, the insoluble protein was pelleted, the supernatant was removed, and a film was cast for mass per volume (w/v%) concentration measurement. Figures 6a and 6c show the w/v% for various boiling times and mass loadings. The dashed lines represent the maximum possible w/v% concentrations based on full theoretical solubility (2, 4, and 8%, respectively). Figures 6b and 6d show the molecular weights of the corresponding solutions used for lyophilization, where 7.5 μg of protein in the solution was run under reducing conditions on a NuPAGE® Novex® 3-8% Tris-Acetate gel (Invitrogen) using HiMark™ pre-stained protein standards (range 30-460 kDa, Invitrogen). Figure 6e represents the data from Figures 6a and 6c in a different manner, showing the concentration of reconstituted silk fibroin solution (mg/mL) and the percentage of solubility.
Figure 7 shows FTIR spectra confirming the transition from amorphous to β-sheet. Films formed from the recovered purified as-prepared (left) or autoclaved (right) were measured before and after 90% methanol treatment for 1 h. Films formed from various boiling times (10-60 MB, top-bottom) are shown in all four sets of graphs, all with different mass loadings (20, 40, 80 mg).
Figure 8 shows the solubility of silk foams formed using only the primary drying cycle after 6 months of dry storage. Silk fibroin solutions generated from various cocoon boiling times (10-60 min boiling, MB) were lyophilized using the (only) primary drying protocol. After completion of lyophilization and 6 months of storage at the designated temperatures (4°C, 22°C, 37°C), 15 mg of the crushed powder of the foam was subsequently resolubilized in 985 μL of deionized water. (a) Assuming negligible solubility loss, 7.5 μg of protein load from each reconstituted solution was run under reducing conditions on a NuPAGE® Novex® 3-8% Tris-acetate gel (Invitrogen) using HighMark™ pre-stained protein standards (range 30-460 kDa, Invitrogen). After centrifugation of the mixture at 1000 rpm, the insoluble protein was pelleted (b), the supernatant was removed, and a film was cast for mass per volume (w/v%) concentration measurement (c). The dotted line represents the maximum possible w/v% concentration based on complete theoretical solubility (1.5%).
Figure 9 shows the solubility of silk foams formed using both primary and secondary drying cycles after 6 months of dry storage. Silk fibroin solutions generated from various cocoon boiling times (10-60 min boiling, MB) were lyophilized using the primary + secondary drying protocol. After completion of the lyophilization cycle and 6 months of storage at the designated temperatures (4°C, 22°C, 37°C), 15 mg of the milled powder of each foam was then resolubilized in 985 μL of deionized water. (a) Assuming negligible solubility loss, 7.5 μg of protein load from each reconstituted solution was run under reducing conditions on a NuPAGE® Novex® 3-8% Tris-acetate gel (Invitrogen) using HighMark™ pre-stained protein standards (range 30-460 kDa, Invitrogen). After centrifugation of the mixture at 1000 rpm, the insoluble protein was pelleted (B), the supernatant was removed, and a film was cast for measurement of the mass per volume (w/v%) concentration (C). The dotted line indicates the maximum possible w/v% concentration based on complete theoretical solubility (1.5%).
Figure 10 shows the solubility of silk films under simulated aging conditions for (a) thick and (b) thin films. Films of various boiling times (also 60 MB film formed from autoclaved solution) were stored at 4 °C, 22 °C, and 37 °C for 6 months before dissolving 40 mg of sample in ultrapure water, compared to day 0. The dotted line indicates the maximum possible w/v% concentration based on full theoretical solubility (4%).
Figure 11 shows the tuned silk recovery based on solution and film design. Using the ELISA kit shown in Table 1, 40 mg of silk films were dissolved in 1 mL (final volume) of PBS, and sample recoveries from the films were normalized to the theoretical maximum load value based on frozen plasma aliquots. Silk solutions formed using different boiling times (10-60 min boiling, MB) were mixed with either plasma or blood, and films were cast in one of two thicknesses (thick vs. thin). Lines indicate significant differences between groups at a significance level of α=0.05 in a two-way ANOVA test. Figures 11a and 11b show fibrinogen recovery from (a) silk-plasma films and (b) silk-blood films. Figures 11c and 11d show C-reactive protein (CRP) recovery from (c) silk-plasma films and (d) silk-blood films.
Figure 12 shows the recovery of bFGF from (a) as-purified silk fibroin solution and (b) autoclaved silk fibroin solution. The addition of PBS improved bFGF regardless of boiling time, concentration, or as-purified versus autoclaved. The solutions were buffered to a final pH of 7.2 by the addition of PBS. bFGF was more stable in the 1% solution than in the 4% solution. The 60 MB silk solution had the highest recovery of bFGF for both the as-purified and autoclaved conditions. The dotted line represents PBS alone as a control condition. The improved bFGF recovery at 37°C for the as-purified silk can be correlated with the absorbance data in Figure 13a in that the gelled 10 MB group had the worst bFGF recovery overall.
Figures 13a and 13b show the gelation results for purified pristine and autoclaved silk fibroin solutions stored at 37°C for one week, respectively. The asterisks in Figure 13a indicate gelation, showing that only the 1% and 4% 10 MB solutions gelled. In Figure 13b, the signal from the 60 MB group is a result of the white staining in the samples, giving false positives for gelation in the absorbance readings, indicating that these samples did not gel. Figure 13 shows that none of the autoclaved samples gelled after one week, and none of the 60 MB group gelled after one week.
Figure 14a shows the gelation status of purified, as-prepared silk fibroin solutions at 4°C, 22°C, and 37°C for a period of 2 weeks, respectively. Asterisks indicate gelation. Samples that are above the baseline without an asterisk are still solutions before gelation. Samples from the 10 MB group tend to gel faster than samples with longer boiling times. The 4% concentration group tends to gel faster than the 1% group. The PBS-containing group gels slower than the unbuffered samples. Figure 14b shows the gelation status of autoclaved silk fibroin solutions at 4°C, 22°C, and 37°C for a period of 2 weeks, respectively. Asterisks indicate gelation. The signals from the 60 MB group give false positives for gelation in the absorbance reading as a result of the white staining in the samples, and these samples did not gel. Samples from the 10 MB group tend to gel faster than those with longer boiling times. The 4% concentration group tends to gel faster than the 1% group. The PBS-containing group gels slower than the unbuffered samples.
Figure 15 is a schematic diagram showing the preparation of thick and thin silk films. Silk fibroin solutions generated from various boiling times (10-60 min boiling, MB) were purified simultaneously, each diluted to 4% wt/v concentration, and 1.5 mL were cast as either thick films (3 x 2.5 cm diameter casting surfaces) or thin films (3 x 7.5 cm diameter casting surfaces). Thin films were produced by spreading the 1.5 mL volume as widely as possible using a pipette tip.
Figure 16 is a bar graph showing the optimization of silk films for complete dissolution. Silk fibroin solutions generated from various boiling times (10-60 min boiling, MB) were cast as either thick films (2.5 cm diameter casting surface) or thin films (7.5 cm diameter casting surface). After air drying overnight, 40 mg coupons of each film were then re-dissolved in 1 mL of deionized water and the wt % of the resulting solutions were measured. The dotted line represents the maximum possible w/v % concentration based on complete theoretical solubility (4%).
Figure 17 is a bar graph showing the optimization of silk foams for complete dissolution. Silk fibroin solutions generated from various cocoon boiling times (10-60 min boiling, MB) were lyophilized using either a primary or primary + secondary drying protocol. After completion of the lyophilization cycle, 15 mg of milled powder of each foam was re-dissolved in 1 mL of deionized water and the wt % of the resulting solution was measured. The dotted line represents the maximum possible w/v % concentration based on complete theoretical solubility (1.5%).
Figure 18 is a histogram showing the solubility of thick silk films under simulated aging conditions. Films at various boiling times (also a 60 MB film formed from autoclaved solutions) were stored at 4°C, 22°C, and 37°C for 6 months prior to dissolving 40 mg of sample in ultrapure water (UPW), as compared to the data from Figure 16 (day 0). The dotted line represents the maximum possible w/v% concentration based on full theoretical solubility (4%).
Figure 19 is a bar graph showing the solubility of silk films under simulated aging conditions. Films of various boiling times (also a 60 MB film formed from autoclaved solutions) were stored at 4°C, 22°C, and 37°C for 6 months prior to dissolving 40 mg of sample in UPW water, as compared to the data from Figure 2 (day 0). The dotted line represents the maximum possible w/v% concentration based on full theoretical solubility (4%).
FIG. 20 is a series of photographs showing film manufacturing and film melting according to one embodiment described herein.
Figure 21 is a series of data graphs showing the measurement of biomarkers by ELISA recovered from blood-stabilized silk films. Silk solutions generated from 30 MB fibroin were formed into films after mixing with whole blood, and then air dried in "thin" samples. Coupons weighing 10 mg or 40 mg were dissolved in 1 mL PBS (1% and 4% w/v loading, respectively) and the concentration conversion is based on the theoretical loading (50 μL:silk ratio in 1 mL blood). The calibration standard curve is shown on the right. Blank (non-loaded) silk films were also used as negative calibration controls.
Figure 22 is a series of data graphs showing the measurement of various biomarkers by Luminex™ recovered from blood- and plasma-stabilized silk films. Silk film coupons weighing 20 mg were dissolved in 1 mL PBS and 25 μL aliquots were run on the Luminex 200™ instrument using three separate CVD biomarker kits. Samples (measured plasma coupons and blood coupons) were taken after 1:200 and 1:2000 dilutions and compared to frozen donor-matched plasma aliquots after 1:2,000 dilution. The internal standard provided absolute quantification and the conversion necessary to calculate theoretical loading of the plasma and blood coupons was based on the above calculations. Blank (unloaded) silk films were also solubilized and used as negative assay controls. All samples were performed in triplicate and are shown as mean ± SD. * Indicates significant difference detected when comparing donors 2 & 3 (p < 0.05 level). Bars represent agreement between load values calculated based on frozen plasma aliquots and matched coupon recoveries.
Figure 23 is a series of bar graphs containing plasma concentrations determined by Luminex and ELISA assay methods. Frozen plasma aliquots were run on the Luminex 200 System and Millipore EMD CVD1 Panel at a 1:200-fold dilution (see Table 5) and on an ELISA kit produced by R&D Systems at a 1:10-fold dilution to compare absolute detection levels (see Table 2). Note that the log axis was used for the CRP assay (left) and the linear axis was used for the PAI-1 assay (right).
Figure 24 is a series of bar graphs showing the percent recovery of various biomarkers, as detected by Luminex™, from blood- and plasma-stabilized silk films at 30 days after exposure to different temperatures. Silk film coupons weighing 20 mg were dissolved in 1 mL PBS and 25 μL aliquots were run on the Luminex 200™ instrument for N=3 donors. Liquid plasma aliquots stored under identical conditions were analyzed similarly. The internal standard provided absolute quantification and the conversion required to calculate theoretical loading of plasma and blood coupons was based on previous calculations (April report). The percent recovery values for the three donors were averaged and expressed as ± SD. The percent recovery defined by the analyte level was divided by the Day 0 plasma level x 100%. Bars indicate significant differences between groups (p < 0.05 level). The dashed line represents the 30-day value stored at -20C. $ indicates coupon values that deviate from 100%. * indicates CV% values in the range of 20-30% from the Luminex system.
Figure 25 is a series of bar graphs showing the percentage recovery of various biomarkers, as detected by Luminex™, from blood- and plasma-stabilized silk films at 4 months after exposure to different temperatures. Silk film coupons weighing 20 mg were dissolved in 1 mL PBS and 25 μL aliquots were run on a Luminex 200™ instrument for N=3 donors. All conditions were the same as at day 30. Liquid plasma aliquots stored under the same conditions were analyzed similarly. Internal standards provided absolute quantification. The % recovery values for the three donors were averaged and expressed as ± SD. The % recovery defined by the level of analyte was divided by the Day 0 plasma level x 100%. The bars indicate significant differences between groups (p < 0.05 level). The dashed line indicates the 30-day value stored at -20C. $ indicates coupon values that deviate from 100%. * Indicates CV% values in the range of 20-30% from the Luminex system.
Figure 26 is a series of photographs showing the formation and solubilization of lyophilized silk compositions. (Top) Silk solutions formed from 20 MB silk fibroin at concentrations ranging from 6% to 1%, with or without addition of TE buffer (EDTA), were lyophilized using primary and secondary drying. The solutions produced shape profiles consistent with their 1.5 mL storage cones. (Bottom) Upon addition of water to the lyophilized solutions and mixing by vortexing, the silk was resolubilized, leaving behind small residual foam fragments, the mass of which is dependent on the mass of the silk load.
Figure 27 is a bar graph showing the recovery of mRNA from lyophilized silk solutions at various concentrations, as measured by the RiboGreen™ assay. Silk solutions formed from 20 MB silk fibroin at concentrations ranging from 2% to 0.05% as well as the non-silk-loaded group (0%) were lyophilized using primary and secondary drying and solubilized in ultrapure water prior to mixing with RiboGreen reagent and fluorometric analysis. The recovered samples were compared to frozen stock solutions of mRNA (-80°C). Duplicate assay readings were performed for all samples. Absolute quantification was performed by comparison to a standard curve generated by ribosomal RNA (provided by the manufacturer), and data represent the mean ± standard deviation of N=3 samples.
Figure 28 is a bar graph showing mRNA recovery from various concentrations of lyophilized silk solutions containing RNAse inhibitor, as measured by the Ribogreen™ assay. Silk solutions formed from 20 MB silk fibroin at concentrations ranging from 2% to 0.05% as well as the non-silk-loaded group (0%) were mixed with SUPERase InTM RNAse inhibitor, lyophilized using primary and secondary drying, and re-solubilized in ultrapure water prior to mixing with Ribogreen reagent and fluorometric analysis. The recovered samples were compared to frozen stock solutions of mRNA (-80°C). Duplicate assay readings were performed for all samples. Absolute quantification was performed by comparison to a standard curve generated by ribosomal RNA (provided by the manufacturer), and data represent the mean ± standard deviation of N=3 samples.
Figure 29 is a series of fluorescence images showing fibroblast transfection using frozen and recovered mRNA with the Stemfect™ transfection kit. The silk solution used in Figure 28 was also mixed with the reagents from the Stemfect™ transfection kit. The transfection reagents were combined with either the frozen stock solution (-80°C) or the recovered mRNA and added to the cells for 12 hours of treatment prior to fluorescence imaging with a GFP filter. White arrows indicate positive GFP expression.
Figure 30 illustrates an overview of one embodiment of silk protein-based stabilization for diagnostics as described herein.

Specific definition

Unless otherwise stated, or implicit from the context, the following terms and phrases have the meanings given below. Unless explicitly stated otherwise, or apparent from the context, the following terms and phrases do not exclude the meanings acquired in the relevant art to which the term or phrase applies. The definitions are provided to assist in describing particular embodiments and are not intended to limit the claimed invention, since the scope of the invention is limited only by the claims. Furthermore, unless the context requires otherwise, singular terms shall include pluralities, and plural terms shall include the singular.

A: The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the term "or" is intended to include "and" unless the context clearly indicates otherwise.

About: Except in the examples or where otherwise stated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with a percentage, the term "about" can mean ± 5% of the stated value. For example, about 100 means 95 to 105.

Ambient: As used herein, “ambient” conditions are ambient conditions without active cooling, heating, etc. Typically, the term refers to room temperature, which generally refers to ordinary room temperature, often 20-25° C. (68-77° F.). In some embodiments, the ambient temperature is between 0° C. and 60° C., between 0° C. and 50° C., or between 0° C. and 40° C. In some embodiments, the ambient temperature is refrigerator temperature (e.g., between 0° C. and 15° C., inclusive). In some embodiments, the ambient temperature is room temperature, e.g., between 20° C. and 35° C., which may vary depending on geographic conditions. For example, room temperature in warm-climate regions, e.g., Africa, may generally be warmer than room temperature in cold-climate regions, e.g., the United States or the United Kingdom. In some embodiments, the storage temperature can be at least about 37° C. or greater than 37° C.

Antigen: As used herein, the term "antigen" refers to a molecule or a portion of a molecule that can be bound by a selective binding agent, such as an antibody, and additionally can be used in an animal to elicit the production of antibodies that can bind to an epitope of the antigen. An antigen can have one or more epitopes. The term "antigen" also refers to a molecule that can be bound by a T cell receptor (TCR) when presented by an antibody, or an MHC molecule. The term "antigen" as used herein also includes a T-cell epitope. An antigen can additionally be recognized by the immune system and/or can induce a humoral immune response and/or a cellular immune response that results in the activation of B- and/or T-lymphocytes. However, this requires, at least in certain cases, that the antigen comprises or is linked to a Th cell epitope and is given as an adjuvant. An antigen can have one or more epitopes (B- and T-epitopes). The specific reaction mentioned above is intended to indicate that the antigen will react favorably with the corresponding antibody or TCR, typically in a very selective manner, and not with a number of other antibodies or TCRs that may be recognized by other antigens. The antigen used herein may also be a mixture of several individual antigens.

Bioactivity: As used herein in connection with an active agent, the term "bioactivity" generally refers to the ability of an active agent to interact with and/or produce an effect on a biological target. For example, bioactivity may include, but is not limited to, stimulating, inhibiting, modulating, inducing a toxic or lethal response in a biological target. The biological target may be a molecule or a cell. For example, bioactivity may refer to the ability of an active agent to modulate the effect/activity of an enzyme, block a receptor, stimulate a receptor, modulate the expression of one or more genes, modulate cell proliferation, modulate cell division, modulate cell morphology, or any combination thereof. In some cases, bioactivity may refer to the ability of a compound to produce a toxic effect on a cell. Exemplary cellular responses include lysis, apoptosis, growth inhibition, and growth promotion; production, secretion, and surface expression of proteins or other molecules of interest by the cell; activation of membrane surface molecules, including receptor activation; transmembrane ion passivation; transcriptional regulation; alteration in cell viability; alteration in cell morphology, alteration in the presence or expression of intracellular components of the cell; Changes in gene expression or transcriptome; changes in the activity of enzymes produced within the cell; and preparation for the presence or expression of ligands and/or receptors (e.g., protein expression and/or binding activity). Methods for assessing different cellular responses are well known to those of ordinary skill in the art, including, for example, Western blots to determine changes in the presence or expression of endogenous proteins of the cell, or microscopic examination to observe cell morphology in response to an active agent, or FISH and/or qPCR to detect and quantify nucleic acid changes. Biological activity can be determined, in some embodiments, for example, by assessing a cellular response.

In relation to an antibody, the term "bioactivity" includes, but is not limited to, epitope or antigen binding affinity, the in vivo and/or in vitro stability of the antibody, e.g., the immunogenic properties of the antibody when administered to a human subject, and/or the ability to antagonize or neutralize the bioactivity of a target molecule in vivo or in vitro. The aforementioned properties or characteristics can be observed or measured using techniques known in the art, including but not limited to scintillation proximity assays, ELISAs, ORIGEN immunoassays (IGEN), fluorescence quenching, fluorescent ELISAs, competitive ELISAs, SPR analysis including but not limited to SPR analysis using a BIAcore biosensor, in vitro and in vivo neutralization assays (see, e.g., International Publication No. WO 2006/062685 ), receptor binding, and immunohistochemistry of tissue sections from different sources including human, primate, or other sources as desired. In relation to immunogens, the term "bioactivity" includes immunogenicity, the definition of which is further elaborated later. In relation to viruses, the term "bioactivity" includes infectivity, the definition of which is further elaborated later. In relation to contrast agents, e.g., dyes, the term "bioactivity" refers to the ability of the contrast agent to enhance the contrast of structures or fluids in the body of a subject when administered to the subject. The bioactivity of a contrast agent also includes, but is not limited to, its ability to influence the response of another molecule under certain conditions and/or to interact with a biological environment.

Cold Chain: The phrase "cold chain" refers to a temperature-controlled supply chain. An intact cold chain is a series of continuous storage and distribution activities that maintain a defined temperature range. It is used to help extend and ensure the shelf life of products such as chemicals, biologics, and pharmaceuticals. Cold chains are common in the food and pharmaceutical industries, and also in the transportation of some chemicals. A typical temperature range for a cold chain in the pharmaceutical industry is 2 to 8°C. In some cases, the specific temperature (and time at temperature) tolerance depends on the actual product being stored and/or transported.

Component: As used herein, the phrase “a component” of a sample refers to a physical, chemical, or biological entity (such as, but not limited to, a protein, peptide, nucleic acid, cell, growth factor, and/or therapeutic agent) present in the sample that can be detected or analyzed.

INCLUDES: Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term "includes" means "including." The abbreviation "e,g," is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "e,g," is synonymous with the term "for example."

Comprising: As used herein, the terms "comprising" or "comprises" are used with respect to compositions, methods, and representative components(s) thereof, and are useful in embodiments that may include elements not specified, whether useful or not.

Reduction: The terms "reduction," "reduced," "reduction," "reduce," or "suppress" are all used herein to generally mean a statistically significant amount of reduction. However, for the avoidance of doubt, "reduced," "reduction," "reduce," or "suppress" means a reduction of at least 10% as compared to a reference level, for example, a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a reduction of less than or equal to 100% (e.g., to the absent level as compared to a reference sample), or any reduction of 10-100% as compared to a reference level.

Essentially: As used interchangeably herein, the terms "essentially" and "substantially" mean a percentage of at least about 60%, or preferably at least about 70% or at least about 80%, or at least about 90%, at least about 95%, at least about 97%, or at least about 99% or more, or any integer from 70% to 100%. In some embodiments, the term "essentially" means a percentage of at least about 90%, at least about 95%, at least about 98%, at least about 99% or more, or any integer from 90% to 100%. In some embodiments, the term "essentially" can include 100%.

Fibroin: As used herein, the term "fibroin" includes silkworm silk fibroin and insect or spider silk proteins. Any type of silk fibroin may be used in accordance with aspects of the present invention (Lucas et al., Adv. Protein Chem 13: 107-242 (1958)). There are many different types of silk produced by a wide variety of species, including, but not limited to: Antheraea mylitta ; Antheraea pernyi; Antheraea yamamai ; Galleria mellonella; Bombyx mori ; Bombyx mandarina ; Galleria mellonella ; Nephila clavipes ; Nephila senegalensis ; Gasteracantha mammosa; Argiope aurantia; Araneus diadematus ; Latrodectus geometricus ; Araneus bicentenarius; Tetragnatha versicolor ; Araneus ventricosus ; Dolomedes tenebrosus; Euagrus chisoseus ; Plectreurys tristis; Argiope trifasciata ; and Nephila madagascariensis . In some embodiments, the fibroin is obtained from a solution containing dissolved silkworm silk or spider silk. Silkworm silk proteins are obtained, for example, from Bombyx mori, and spider silk is obtained from Nephila clavipes. Other silks include transgenic silks, such as those from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants and variants thereof, and genetically engineered silks (recombinant silks). See, for example, WO 97/08315 and U.S. Pat. No. 5,245,012, the contents of which are both incorporated herein by reference in their entirety. In some embodiments, silk fibroin may be derived from other sources, such as spiders, other silkworms, bees, synthetic silk-like peptides, and biotechnological variants thereof. In some embodiments, silk fibroin may be extracted from the glands of silkworms or transgenic silkworms. See, for example, WO2007/098951, the contents of which are incorporated herein by reference in their entirety. Although different species of silk-producing organisms and different types of silk have different amino acid compositions, the various silk proteins share certain structural features. A common pattern in the silk fibroin structure is a sequence of amino acids that is generally characterized by alternating glycine and alanine, or alanine alone. Such arrangements allow the fibroin structure to self-assemble into a beta-sheet conformation. These "alanine-rich" and "glycine-rich" hydrophobic blocks are typically separated by segments of amino acids having bulky side groups (e.g., hydrophilic spacers). In some embodiments, the core repeat sequence of the hydrophobic block of fibroin can be represented by the following amino acid sequences and/or formulas: (GAGAGS)5-15 (SEQ ID NO: 1); (GX)5-15 (X=V, I, A) (SEQ ID NO: 2); GAAS (SEQ ID NO: 3); (S1-2A11-13) (SEQ ID NO: 4); GX1-4 GGX (SEQ ID NO: 5); GGGX (X=A, S, Y, R, DV, W, R, D) (SEQ ID NO: 6); (S1-2A1-4)1-2 (SEQ ID NO: 7); GLGGLG (SEQ ID NO: 8); GXGGXG (X=L, I, V, P) (SEQ ID NO: 9); GPX (X=L, Y, I); (GP(GGX)1-4 Y)n (X=Y, V, S, A) (SEQ ID NO: 10); GRGGAn (SEQ ID NO: 11); GGXn (X=A, T, V, S); GAG(A)6-7GGA (SEQ ID NO: 12); and GGX GX GXX (X=Q, Y, L, A, S, R) (SEQ ID NO: 13). In some embodiments, the fibroin peptide can contain multiple hydrophobic blocks within the peptide, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 hydrophobic blocks. In some embodiments, the fibroin peptide can contain 4 to 17 hydrophobic blocks. In some embodiments of the invention, the fibroin peptide comprises at least one hydrophilic spacer sequence (“hydrophilic block”) that is about 4-50 amino acids in length. Non-limiting examples of hydrophilic spacer sequences include: TGSSGFGPYVNGGYSG (SEQ ID NO: 14); YEYAWSSE (SEQ ID NO: 15); SDFGTGS (SEQ ID NO: 16); RRAGYDR (SEQ ID NO: 17); EVIVIDDR (SEQ ID NO: 18); TTIIEDLDITIDGADGPI (SEQ ID NO: 19) and TISEELTI (SEQ ID NO: 20). In certain embodiments, the fibroin peptide can contain a hydrophilic spacer sequence that is a derivative of any one of the exemplary spacer sequences listed above. Such derivatives are at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of the hydrophilic spacer sequences. In some embodiments, the fibroin peptides suitable for the present invention do not contain spacers. Silk is generally a fibrous protein and is characterized by modular units, highly repetitive proteins, linked together to form high molecular weights. These modular units, or domains, each with a specific amino acid sequence and chemistry are thought to provide specific functions. For example, sequence motifs such as poly-alanine (polyA) and poly-alanine-glycine (poly-AG) tend to form beta sheets; the GXX motif contributes to the formation of a 31-helix; the GXG motif provides rigidity; and, GPGXX (SEQ ID NO: 22) contributes to the formation of beta helices. These are examples of different components in various silk structures whose location and arrangement are relevant to the ultimate material properties of the silk material (reviewed in Omenetto and Kaplan (2010) Science 329: 528-531). See also WO 2011/130335 (PCT/US2011/032195), the contents of which are incorporated herein by reference.

Increase: The terms "increased," "increase," or "enhance," or "activate," are all used herein to mean an increase, generally a statistically significant amount; for the avoidance of any doubt, the terms "increased," "increase," or "enhance," or "activate" mean an increase of at least 10% over a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to (and including a 100%) over a reference level, or any increase of from 10 to 100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase from 2-fold to 10-fold or more.

Inhibition: As used herein, the term "inhibit" means preventing an event from occurring, delaying the occurrence of an event, and/or reducing the extent or likelihood of an event.

Maintenance: The terms "maintaining," "maintain," and "maintaining," as used herein, when referring to a composition or an activator, mean maintaining, continuing, or containing the activity of one or more activators of the silk fibroin matrix when the activator is placed under certain conditions. In some embodiments, the one or more activators distributed in the silk fibroin matrix retains at least about 30% of its original activity, including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more of its original activity.

Nanopattern: As used herein, the term "nanopattern" or "nanopatterned" refers to a small patterning provided in a silk fibroin substrate matrix, such as a film or foam, or a composition comprising such a silk fibroin substrate matrix. The patterning having structural features of a size generally can be suitably measured in the nanoscale (i.e., on the order of 10 ^-9 meters), for example, in the range of 1 nanometer to a millimeter.

Nucleic Acid: As used herein, the term "nucleic acid" or "oligonucleotide" or grammatical equivalents herein mean at least two nucleotides (including analogs or derivatives thereof) covalently linked to each other. Exemplary nucleic acids include, but are not limited to, polynucleotides, oligonucleotides, genes, genes including regulatory and terminal regions, self-replicating systems such as viruses or plasmid DNA, genomic DNA, cDNA, mRNA, pre-mRNA, single-stranded and double-stranded siRNA and other RNA interference reagents (RNAi reagents or iRNA reagents), shRNA (short hairpin RNA), antisense oligonucleotides, antisense oligonucleotides, aptamers, ribozymes, microRNA (miRNA), pre-miRNA, and modified RNA (e.g., locked nucleic acids). A nucleic acid can be single-stranded or double-stranded. The nucleic acid may be DNA, RNA or a hybrid wherein the nucleic acid contains any combination of deoxyribo- and ribonucleotides, and any combination of uracil, adenine, thymine, cytosine and guanine. Nucleic acids may contain one or more backbone modifications, such as phosphoramide (see Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970)), phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), or peptide nucleic acid linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); and Nielsen, Nature, 365:566 (1993)], the entire contents of which are incorporated herein by reference. Nucleic acids may also include modifications of the sugar moiety of the nucleobase and/or nucleotide. Exemplary sugar modifications at the sugar moiety include 2'-OH substituted with halogen (e.g., fluoro), O-methyl, O-methoxyethyl, NH ₂ , SH, and S-methyl.

Polyethylene Glycol: "PEG" means an ethylene glycol polymer containing from about 20 to about 2,000,000 linked monomers, typically from about 50 to about 1,000 linked monomers, usually from about 100 to about 300. Polyethylene glycol includes PEGs containing various numbers of linked monomers, for example, PEG20, PEG30, PEG40, PEG60, PEG80, PEG100, PEG115, PEG200, PEG 300, PEG400, PEG500, PEG600, PEG1000, PEG1500, PEG2000, PEG3350, PEG4000, PEG4600, PEG5000, PEG6000, PEG8000, PEG11000, PEG12000, PEG2000000 and mixtures of any of these.

Prevention: As used herein, the term "preventing" when used to refer to the action of an agent on a course (e.g., pathogenesis, disease progression, etc.) means that when the agent (e.g., therapeutic agent) is administered prior to the development of one or more symptoms or traits associated with the course, it lessens the severity of and/or delays the onset of such course.

Ready-to-use: The phrase "ready-to-use" in "ready-to-use formulation" refers to a composition or product that requires no additional processing (e.g., a manufacturing process) prior to use by the end user. In some embodiments, the ready-to-use formulation comprises an injectable formulation (i.e., an injection). As used herein, ready-to-use formulations encompass concentrated formulations, such as concentrated solution formulations, that are designed to be diluted prior to administration.

REFERENCE: The term "reference" as used herein refers to, for example, a reference composition or set of conditions, etc. A person of ordinary skill in the art will understand from the context what an appropriate "reference" is in a particular situation. Typically, a "reference" shares sufficient similarity with a composition of interest or set of conditions to allow for a meaningful comparison. In many embodiments, a "reference composition" herein is a conventional silk-fibroin composition (e.g., one that is not a low molecular weight silk fibroin composition).

Short hairpin RNA: As used herein, the term "shRNA" refers to short hairpin RNAs that function like RNAi and/or siRNA species, but differ in that shRNA species have a double-stranded hairpin-like structure for increased stability. As used herein, the term "RNAi" refers to interfering RNA, or an RNA interference molecule, which is a nucleic acid molecule or analog thereof, e.g., an RNA-based molecule, that inhibits gene expression. RNAi refers to a means of selective post-transcriptional gene silencing. RNAi can result in the destruction of a specific mRNA, or in interference with the processing or translation of an RNA, e.g., an mRNA.

Short interfering RNA: The term "short interfering RNA" (siRNA) (also referred to herein as "small interfering RNA") is defined as an agent that functions to inhibit the expression of a target gene, for example, by RNAi. siRNAs can be chemically synthesized, produced by in vitro transcription, or produced within a host cell. siRNA molecules can also be produced by cleavage of double-stranded RNA, where one strand is identical to the message being inactivated. The term "siRNA" refers to a small inhibitory RNA duplex that induces the RNA interference (RNAi) pathway. These molecules can vary in length (typically 18-30 base pairs) and can have varying degrees of complementarity to their target mRNA on the antisense strand. Some, but not all, siRNAs have unpaired overhanging bases at the 5' or 3' end of the sense strand and/or the antisense strand. The term "siRNA" includes a duplex of two individual strands, as well as a single strand capable of forming a hairpin structure comprising a duplex region.

Solution: The term "solution" broadly refers to a homogeneous mixture consisting of one phase. Typically, a solution comprises a solute or solutes dissolved in a solvent or solvents. This is characterized by the fact that the properties of the mixture (e.g., concentration, temperature, and density) can be distributed uniformly throughout the volume. Thus, in the context of the present application, "silk fibroin solution" refers to silk fibroin protein in a soluble form dissolved in a solvent, such as water. In some embodiments, the silk fibroin solution may be prepared from a solid silk fibroin material (i.e., a silk matrix), such as a silk film or other scaffold. Typically, the solid silk fibroin material is reconstituted in an aqueous solution, such as water and a buffer. It should be noted that non-homogeneous liquid mixtures, such as colloids, suspensions, and emulsions, are not considered solutions. To give just one example, silk fibroin microspheres or particles suspended in a solution do not, by themselves, constitute a silk fibroin solution.

Stabilization: As used herein, "stabilization" of an agent by soluble silk fibroin refers to any effect of the silk fibroin polypeptide that supports, promotes, facilitates, and/or maintains the structural integrity (e.g., conformation) and corresponding function or activity of the agent such that the agent is less susceptible to degradation, misfolding, denaturation, aggregation, and/or inactivation. A more detailed discussion of the stabilizing effect of a silk fibroin matrix is provided in PCT/US12/34643 (filed April 23, 2012) and Zhang et al. (2012) Proc. Nat'l. Acad. Sci. U.S.A. 109(30): 11981-6, the contents of which are incorporated herein by reference in their entireties. According to various aspects of the embodiments described herein, at least one property of one or more components of a biological sample can be stabilized within the low molecular weight silk fibroin composition for any period of time. As used herein, the terms "stabilize," "stabilizing," or "stabilization" refer to the partial or complete retention or retention of one or more properties (e.g., activity, integrity, and/or amount) of a component of a biological sample in a low molecular weight silk-based material over a period of time. In some embodiments, with respect to stabilizing an activity, at least about 30% or more (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of the component originally present in the biological sample can retain at least partial or complete activity over a period of time. In other words, the component can retain at least about 30% or more (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of its original activity over a period of time. With respect to stabilization of integrity, at least about 30% (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of a component originally present in the biological sample can be retained over a period of time. With respect to stabilization of quantity, at least about 30% (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of a component originally present in the biological sample can be retained over a period of time, regardless of whether the component is active or inactive, or intact or non-intact. As used herein, the terms "original" or "original" as used in reference to the original presence or original activity of a component refer to the level of the component as measured immediately after the biological sample is obtained from the subject, or alternatively immediately before or after the biological sample is mixed or incorporated into the silk-based material. In some embodiments, the original presence or original activity of a component refers to the level of the component in a control, e.g., a control stabilized by a non-silk approach, such as a frozen control.

Statistically significant: The term "statistically significant" or "significantly" refers to statistical significance, which is usually less than or more than a reference level of two standard deviations (2SD). The term refers to statistical evidence that a difference exists. It is defined as the probability of making a decision against the null hypothesis when the null hypothesis is actually true.

Subject: As used herein, "subject" means a human or an animal. Typically, an animal is a vertebrate, such as a primate, rodent, domestic or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys and macaques, e.g., rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cats, canine species, e.g., dogs, foxes, wolves, bird species, e.g., chickens, emus, ostriches and fish, e.g., trout, catfish and salmon. A patient or subject includes any subset of the foregoing, e.g., all or one or more populations or species, excluding humans, primates or rodents. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms "patient" and "subject" are used interchangeably herein.

Susceptible: As used herein, the term "susceptible" means having an increased risk and/or predisposition (typically due to genetic predisposition, environmental factors, personal history, or a combination thereof) to something, such as a disease, disorder or condition (such as cancer) beyond what is observed in the general population. This term takes into account that an individual's "susceptibility" to a condition is in no way diagnostic of the condition.

Tube: The term "tube" herein refers to an elongated shaft having a lumen. The tube may typically be an elongated hollow cylinder, but may also be a hollow shaft of other cross-sectional shapes.

While preferred embodiments have been described and illustrated in detail herein, it will be apparent to those skilled in the art that various modifications, additions, substitutions, etc. may be made therein without departing from the spirit of the invention and are therefore considered within the scope of the invention as defined in the claims that follow. Furthermore, to the extent already indicated, it will be appreciated by those skilled in the art that any one of the various embodiments described and illustrated herein may be further modified by incorporating features shown in any of the other embodiments disclosed herein.

The present disclosure is further illustrated by the following examples, which should not be construed as limiting. These examples are provided for illustrative purposes only and are not intended to limit the present disclosure in any manner or in any aspect. The following examples do not limit the present disclosure in any way.

Detailed description of specific embodiments

It is to be understood that the present invention is not limited to the particular methodologies, protocols, and reagents described herein, and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms include plural references and vice versa, unless the context clearly dictates otherwise. It should be understood that, other than in the examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are modified in all instances by the term "about."

All identified patents and other publications are expressly incorporated herein by reference for the purpose of describing and disclosing, by way of example, the methodologies described in such publications that can be used with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the inventors have no right to antedate such disclosure by virtue of prior invention or for any other reason. Any statement as to the date of these documents, or any representation as to the contents of these documents, is based on information available to the applicant and does not constitute an admission as to the accuracy of the date or contents of these documents.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any known methods, devices, and materials can be used in the practice or testing of the present invention, the methods, devices, and materials in this regard are described herein.

low molecular weight composition

One aspect provided herein relates to a low molecular weight silk fibroin composition. According to one aspect of the present invention, the low molecular weight silk fibroin composition comprises a population of silk fibroin fragments having a molecular weight range, wherein no more than 15% of the total number of silk fibroin fragments in the population have a molecular weight greater than 200 kDa, and no more than 50% of the total number of silk fibroin fragments in the population have a molecular weight within a particular range of from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa.

In other words, in some embodiments, the low molecular weight silk fibroin composition comprises a population of silk fibroin fragments having a molecular weight range, characterized in that no more than 15% of the total moles of silk fibroin fragments in the population have a molecular weight greater than 200 kDa, and no more than 50% of the total moles of silk fibroin fragments in the population have a molecular weight within a particular range of from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa.

In some embodiments, the low molecular weight silk fibroin composition comprises a population of silk fibroin fragments having a molecular weight range, wherein no more than 15% of the total weight of the silk fibroin fragments in the population have a molecular weight greater than 200 kDa, and no more than 50% of the total weight of the silk fibroin fragments in the population have a molecular weight within a particular range of from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa.

As used herein, the phrase "silk fibroin fragment" refers to a peptide chain or polypeptide having an amino acid sequence corresponding to a fragment derived from silk fibroin protein or a variant thereof. In the context of the present disclosure, silk fibroin fragments generally refer to silk fibroin peptide chains or polypeptides that are smaller than naturally occurring full-length silk fibroin, wherein one or more of the silk fibroin fragments in the population or composition have a size of 300 kDa or less, 250 kDa or less, 200 kDa or less, 175 kDa or less, 150 kDa or less, 120 kDa or less, 100 kDa or less, 90 kDa or less, 80 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, 15 kDa or less, 10 kDa or less, 9 kDa or less, 8 kDa or less, 7 kDa or less, 6 kDa or less, 5 kDa or less, 4 kDa or less, 3.5 kDa or less, etc.

In some embodiments, a "composition comprising a population of silk fibroin fragments" encompasses a composition comprising non-fragmented (i.e., full-length) silk fibroin polypeptides in addition to shorter fragments of silk fibroin polypeptides. The silk fibroin fragments described herein may be produced as recombinant proteins or may be derived or isolated (e.g., purified) from natural silk fibroin proteins or silkworm cocoons.

In some embodiments, the silk fibroin fragments can be derived by refining silkworm cocoons under specified conditions to produce silk fibroin fragments having a desired range of molecular weights.

In some embodiments, the silk fibroin fragments can be derived from refining silkworm cocoons at or near atmospheric boiling temperature for at least about 60 minutes or more (e.g., including at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least about 120 minutes or more). As used herein, the term "atmospheric boiling temperature" refers to the temperature at which a liquid boils under atmospheric pressure.

In some embodiments, the silk fibroin fragments can be produced by refining silkworm cocoons in an aqueous solution at about 90° C. to about 110° C. for at least 60 minutes or more, including at least 70 minutes or more. In some embodiments, the silk fibroin fragments can be derived from refining silkworm cocoons at or below ambient boiling temperature for a longer period of time, such as greater than 60 minutes or more, including greater than 70 minutes, greater than 80 minutes, greater than 90 minutes, greater than 100 minutes, greater than 110 minutes, greater than 120 minutes, greater than 130 minutes, greater than 140 minutes, greater than 150 minutes, or more.

Without wishing to be bound by theory, the silk fibroin fragments can be produced by refining silkworm cocoons at a temperature of about 70° C. for at least 60 minutes or more (e.g., including at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes or more). In some embodiments, the silk fibroin fragments can be produced by a process comprising refining silkworm cocoons under specified conditions and further treating the resulting silk fibroin solution at elevated temperature and/or pressure. For example, the silk fibroin fragments can be produced by a process comprising refining silkworm cocoons at about a boiling temperature for about 10 minutes and then treating the resulting silk fibroin solution at elevated temperature and/or pressure (e.g., autoclaving).

In one embodiment, an example of a low molecular weight silk fibroin composition can be produced as follows: the silk fibroin preparation is carried out in an amount sufficient to achieve fragmentation of the silk fibroin polypeptide, characterized in that at least 50 wt % of the total silk fibroin present in the silk fibroin preparation exhibits a reduced weight, i.e., smaller weight (e.g., silk fibroin fragments), than full-length silk fibroin, as measured, e.g., by SDS gel electrophoresis. For example, for a silk fibroin preparation containing a total of 1.0 gram full-length silk fibroin as starting material, after the heating and/or boiling step, at least 0.5 gram of the silk fibroin is now in a reduced form (smaller fragments) compared to the starting material (e.g., full-length polypeptide). As a result, the average weight of silk fibroin polypeptides within the preparation will decrease upon formation of low molecular weight silk fibroin compositions.

In some embodiments, the silk fibroin fragments in the low molecular weight silk fibroin compositions described herein can be substantially free of sericin. In some embodiments, the silk fibroin fragments can contain less than or equal to 5 wt % sericin of the total weight of the silk protein composition. For example, the silk fibroin fragments can contain less than or equal to 4 wt %, less than or equal to 3 wt %, less than or equal to 2 wt %, less than or equal to 1 wt %, or less than or equal to 0.5 wt % sericin of the total weight of the silk protein composition.

In some embodiments, the silk fibroin fragment can be modified. For example, in some embodiments, the silk fibroin fragment can be modified to include a functional group. In some embodiments, the silk fibroin fragment can be covalently or non-covalently linked or fused to an agent, including but not limited to a peptide, a protein, a nucleic acid molecule, an imaging agent, a therapeutic agent, a target binding ligand, a cell binding ligand, an amphiphilic peptide, or any combination thereof. In some embodiments, the amino acid sequence of the silk fibroin fragment can include a cell binding ligand and/or an amphiphilic peptide. In one embodiment, the amino acid sequence of the silk fibroin fragment can include an RGD sequence.

In embodiments of the low molecular weight silk fibroin compositions described herein, no more than 15% of the total number (or total moles) or total weight of the silk fibroin fragments in the population have a molecular weight greater than 200 kDa. As used herein, the phrase "greater than 200 kDa" refers to a molecular weight of silk fibroin fragments greater than 200 kDa. In some embodiments, the phrase "greater than 200 kDa" can encompass a molecular weight of silk fibroin fragments that is about 200 kDa. In some embodiments, the silk fibroin fragments having a molecular weight greater than 200 kDa are present in the population in an amount less than or equal to 10% of the total number (or total moles) or total weight of the silk fibroin (e.g., less than or equal to 9%, less than or equal to 8%, less than or equal to 7%, less than or equal to 6%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, or less than or equal to 1% of the total number (or total moles) or total weight of the silk fibroin). In one embodiment, the low molecular weight silk fibroin composition is substantially free of silk fibroin fragments having a molecular weight greater than 200 kDa.

In the low molecular weight silk fibroin compositions described herein, at least 50% of the total number (or total moles) or total weight of the silk fibroin fragments have a molecular weight within a particular range of from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa. In some embodiments, at least 50% (including, for example, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more) of the total number (or total moles) or total weight of the silk fibroin fragments can have a molecular weight within the particular range. In one embodiment, the low molecular weight silk fibroin composition can have a population of silk fibroin fragments having a molecular weight of substantially from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa.

The molecular weight distribution of the silk fibroin fragments present in at least 50% of the total number (or total moles) or total weight of the silk fibroin fragments in the population can vary from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa. In some embodiments, the molecular weight distribution of at least 50% of the total number (or total moles) or total weight of the silk fibroin fragments can have a lower limit of 3.5 kDa or more, but less than 120 kDa. For example, the lower end of the specified range can be 3.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 115 kDa. In some embodiments, the specified range of molecular weight distribution of at least about 50% of the total number (or total moles) or total weight of the silk fibroin fragments can have an upper end of 10 kDa or more (including up to 120 kDa). By way of example only, the upper limit of the specified range can be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 kDa. Examples of specified ranges include, but are not limited to: (i) between about 5 and 120 kDa; (ii) between about 10 and 120 kDa; (iii) between about 15 and 120 kDa; (iv) between 20 and 120 kDa; (v) between 20 and 110 kDa; (vi) between about 20 and 100 kDa; (vii) between about 20 and 90 kDa; (viii) between about 20 and 80 kDa; (ix) between about 30 and 120 kDa; (x) between about 30 and 100 kDa; (xi) between about 30 and 90 kDa; (xii) between about 30 and 80 kDa; (xiii) between about 40 and 120 kDa; (xiv) between about 40 and 110 kDa; (xv) between about 40 and 100 kDa; (xvi) between about 40 and 90 kDa; and (xvii) between about 40 and 80 kDa.

Silk fibroin fragments having a molecular weight distribution within a specified range as described above can exhibit a continuous or discrete molecular weight distribution. As used herein, the term "continuously molecular weight distribution" refers to a distribution of molecular weights having any subrange between a specified range. As used herein, the term "discretely molecular weight distribution" refers to a distribution of molecular weights having a specified subrange between a specified range. By way of example only, silk fibroin fragments having a discrete molecular weight distribution within a specified range of about 3.5 kDa to about 120 kDa, or about 5 kDa to about 125 kDa, refers to a population of silk fibroin fragments wherein some of the silk fibroin fragments have a molecular weight from about 3.5 kDa to about 10 kDa, but at least some or the remainder of the silk fibroin fragments have a molecular weight from about 110 kDa to about 120 kDa.

Accordingly, in some embodiments, at least about 50% or more of the total number (or total moles) or total weight of the silk fibroin fragments in the population having a molecular weight within a particular range of about 3.5 kDa to about 120 kDa, or about 5 kDa to about 125 kDa can be characterized as a population of silk fibroin fragments, wherein at least about 50% or more of the total number (or total moles) or total weight of the silk fibroin fragments in the population having a molecular weight within a particular range are selected from the following subranges (i)-(x), (i) silk fibroin having a molecular weight distribution of 20 kDa to 30 kDa; (ii) silk fibroin having a molecular weight distribution of 30 kDa to 40 kDa; (iii) silk fibroin having a molecular weight distribution of 40 kDa to 50 kDa; (iv) silk fibroin having a molecular weight distribution of 50 kDa to 60 kDa; (v) silk fibroin having a molecular weight distribution of 60 kDa to 70 kDa; (vi) silk fibroin having a molecular weight distribution of 70 kDa to 80 kDa; (vii) silk fibroin having a molecular weight distribution of 80 kDa to 90 kDa; (viii) silk fibroin having a molecular weight distribution of 90 kDa to 100 kDa; (ix) silk fibroin having a molecular weight distribution of 100 kDa to 110 kDa; and (x) one or more of silk fibroin having a molecular weight distribution of 110 kDa to 120 kDa (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

The amount of silk fibroin fragments having molecular weight subranges (i) to (x) can vary from 0% to 100% of the total number (or moles) or total weight of all silk fibroin fragments in the composition described herein, provided that the combined weight of the silk fibroin fragments having molecular weight subranges (i) to (x) constitutes at least 50% or more of the total number (or moles) or total weight of all silk fibroin fragments in the composition described herein. Accordingly, the low molecular weight silk fibroin compositions described herein can be configured to have any combination of silk fibroin fragments having molecular weight subranges (i) to (x). In some embodiments, the low molecular weight silk fibroin composition can have silk fibroin fragments corresponding to one particular molecular weight subrange as defined herein. In other embodiments, the low molecular weight silk fibroin composition can have a mixture of silk fibroin fragments corresponding to two or more particular molecular weight subranges as defined herein.

In some embodiments, the ratio of silk fibroin fragments having a molecular weight of 76 kDa to silk fibroin fragments having a molecular weight of 18 kDa is not greater than 5:1 to 1.5:1. Accordingly, in some embodiments, the low molecular weight silk fibroin composition comprises a population of silk fibroin fragments having a molecular weight range characterized by having a molecular weight within a particular range of from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa, wherein no more than 15% of the total number (or moles) or total weight of the silk fibroin fragments in the population have a molecular weight greater than 200 kDa, and wherein at least 50% of the total number (or moles) or total weight of the silk fibroin fragments in the population have a molecular weight of 76 kDa to silk fibroin fragments having a molecular weight of 18 kDa is not greater than 5:1 to 1.5:1.

In some embodiments, the low molecular weight silk fibroin composition can further comprise no more than 35% of the total number (or moles) or total weight of silk fibroin fragments having a molecular weight distribution of 120 kDa to 200 kDa. For example, in some embodiments, less than 35% (including no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 3%, no more than 1%) of the total number (or moles) or total weight of silk fibroin fragments having a molecular weight distribution of 120 kDa to 200 kDa can be present in the compositions described herein. In one embodiment, the compositions described herein can be substantially free of silk fibroin fragments having a molecular weight distribution of 120 kDa to 200 kDa.

In some embodiments, wherein the composition comprises silk fibroin fragments having a molecular weight distribution of 120 kDa to 200 kDa, the silk fibroin fragments are selected from the group consisting of subranges (xi)-(xviii), (xi) silk fibroin having a molecular weight distribution of 120 kDa to 130 kDa; (xii) silk fibroin having a molecular weight distribution of 130 kDa to 140 kDa; (xiii) silk fibroin having a molecular weight distribution of 140 kDa to 150 kDa; (xiv) silk fibroin having a molecular weight distribution of 150 kDa to 160 kDa; (xv) silk fibroin having a molecular weight distribution of 160 kDa to 170 kDa; (xvi) silk fibroin having a molecular weight distribution of 170 kDa to 180 kDa; (xvii) silk fibroin having a molecular weight distribution of 180 kDa to 190 kDa; and (xviii) silk fibroin having a molecular weight distribution of 190 kDa to 200 kDa (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

In some embodiments, the composition described herein can be superior in at least one or more silk fibroin fragments within the specific subranges (i)-(xviii) defined herein compared to a reference silk fibroin composition. In some embodiments, the reference silk fibroin composition can be a composition or mixture produced by refining silkworm cocoons at ambient boiling temperature for about 60 minutes. In one embodiment, the reference silk fibroin composition can be a composition or mixture produced by refining silkworm cocoons in an aqueous sodium carbonate solution at ambient boiling temperature for about 60 minutes. In some embodiments, the composition described herein can be superior in at least two or more silk fibroin fragments (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) within the specific subranges (i)-(xviii) defined herein compared to a reference silk fibroin composition. In some embodiments, the at least one silk fibroin fragment can be present in the composition by at least about 10% or more (e.g., including at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more) as compared to a reference silk fibroin composition. In some embodiments, the at least one silk fibroin fragment can be present in the composition by at least about 1.1-fold or more (e.g., including at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold or more) as compared to a reference silk fibroin composition.

According to various embodiments described herein, the low molecular weight silk fibroin compositions are distinguished from so-called "hydrolyzed silk". Hydrolyzed silk is generally produced by hydrolyzing or breaking down silk proteins into smaller protein chains, such as those having a molecular weight of less than 1 kDa and/or constituent amino acids, such as glycine, alanine and serine. Accordingly, the term "hydrolyzed silk" as used herein refers to silk peptide chains or amino acids having a molecular weight of less than 2 kDa, less than 1 kDa, less than 500 Da or less. In other words, the hydrolyzed silk is generally free of silk fibroin peptide chains having a molecular weight of at least 3.5 kDa or more (e.g., at least 5 kDa, at least 10 kDa, at least 15 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa or more).

The molecular weight of the silk fibroin fragments described herein can be determined by any method known in the art, including but not limited to, SDS-PAGE gel, size exclusion gel chromatography, mass spectrometry, or any combination thereof. In some embodiments, the molecular weight of the silk fibroin fragments referenced herein in the low molecular weight silk fibroin compositions described herein can be determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). By way of example only, for each low molecular weight fibroin composition, an appropriate amount of the composition or silk fibroin protein (e.g., about 7.5 μg) can be prepared in solution and then loaded into a gel, such as a 3-8% Tris-acetate gel (e.g., NuPAGE® Novex® 3-8% Tris-acetate gel obtained from Invitrogen). Protein standards or ladders are also loaded into the gel to provide reference molecular weights. Examples of protein standards can include, but are not limited to, HighMark™ Pre-Stained Protein Standards (30-460 kDa range, Invitrogen) and Mark12™ Protein Standards (30-200 kDa range, Invitrogen). The gel can be run under reducing conditions according to the manufacturer's instructions. For example, in one embodiment, the gel can be run at about -200 V for about 45 minutes and then stained to visualize the protein bands. In some embodiments, the gel can be stained with a colloidal blue stain kit, for example, obtained from Invitrogen. The molecular weight distribution of the silk fibroin fragments present in the low molecular weight silk fibroin composition can then be quantified using any recognized technical method, for example, by taking densitometric measurements of the protein bands along the length of a sample lane of the gel. In one embodiment, ImageJ (NIH, Bethesda, MD) can be used to determine densitometric measurements of protein bands on a gel. In one embodiment, the “Gel Analyzer” tool within the ImageJ software can be used to perform densitometric analysis.

Without limitation, the molecular weight used herein can be peak average molecular weight (Mp), number average molecular weight (Mn), or weight average molecular weight (Mw).

In some embodiments, the low molecular weight silk fibroin composition can further comprise at least one activator, examples of which are described below. The activator can be dispersed into the compositions described herein by any method known in the art. For example, the activator can be dispersed homogeneously or heterogeneously (e.g., forming a gradient of activator) into the compositions described herein (see U.S. Application Publication No. US20070212730 A1, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the agent included in the low molecular weight silk fibroin composition can be stored in the composition, and/or released or recovered from the composition, whether in liquid or solid form. In some embodiments, the agent included can be analyzed, for example, before, during, or after such release or recovery.

Low molecular weight solutions:

In some embodiments, the low molecular weight silk fibroin compositions provided herein can be a solution (optionally comprising an agent, such as an agent to be stabilized and/or analyzed as described herein). Accordingly, in another aspect, provided herein is an aqueous silk fibroin solution comprising one or more embodiments of the low molecular weight silk fibroin compositions described herein. In some embodiments, the aqueous silk fibroin solution can be formulated in water. In some embodiments, the aqueous silk fibroin solution can be formulated in a buffered solution. Examples of buffered solutions include, but are not limited to, a phosphate buffered solution.

The silk fibroin can be present in the solution at any concentration suitable for the purpose of, for example, the injectability of, the silk fibroin solution. In some embodiments, the aqueous silk fibroin solution can have silk fibroin at a concentration of about 0.1% wt/v to about 90% wt/v, about 0.1% wt/v to about 75% wt/v, or about 0.1% wt/v to about 50% wt/v. In some embodiments, the aqueous silk fibroin solution can have silk fibroin at a concentration of about 0.1% wt/v to about 10% wt/v, about 0.1% wt/v to about 5% wt/v, about 0.1% wt/v to about 2% wt/v, or about 0.1% wt/v to about 1% wt/v. In some embodiments, the water-soluble silk fibroin solution has silk fibroin at a concentration of about 10% wt/v to about 50% wt/v, about 20% wt/v to about 50% wt/v, about 25% wt/v to about 50% wt/v, or about 30% wt/v to about 50% wt/v.

In some embodiments, the aqueous solution can remain stable under certain conditions for at least about 3 days or more (e.g., including at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks or more). The certain conditions can be characterized by one or more of environmental parameters including, but not limited to, temperature, light, humidity, temperature, pressure, and any combination thereof. In some embodiments, the aqueous silk solution can remain stable at a temperature from about room temperature to at least about 37° C. or more for at least about 3 days or more (e.g., including at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks or more). In some embodiments, the aqueous solution does not gel upon exposure of the aqueous silk fibroin solution to particular conditions for at least about 3 days or more (e.g., at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks or more). In some embodiments, the aqueous silk solution does not gel upon exposure of the aqueous silk fibroin solution to a temperature at least room temperature or higher (e.g., at least about 15° C., at least about 20° C., at least about 25° C., at least about 30° C., at least about 35° C., at least about 40° C. or more) for at least about 3 days or more (e.g., at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks or more).

In some embodiments, the low molecular weight silk fibroin solutions described herein gel more slowly than a reference silk fibroin solution as defined herein. In some embodiments, the low molecular weight silk fibroin solutions described herein gel more slowly than a conventional silk fibroin solution.

As used herein, the term "remains stable" refers to a material property of a water-soluble silk fibroin solution that remains substantially the same upon exposure to specified conditions for a period of time. Examples of material properties may include, but are not limited to, viscosity, particle size, opacity, and any combination thereof. In some embodiments, a stable water-soluble silk solution can be characterized by no substantial change in viscosity upon exposure to specified conditions for a period of time. In some embodiments, a stable water-soluble silk solution can be characterized as a homogeneous solution, as defined herein. For example, a homogeneous water-soluble silk fibroin solution can be characterized as a water-soluble silk fibroin solution that is substantially free of silk fibroin aggregates (e.g., insoluble silk fibroin fragments, silk fibroin particles and/or clusters visible to the naked eye) upon exposure to specified conditions for a period of time. Examples of silk fibroin aggregates can include, but are not limited to, full-length silk fibroin molecules, longer silk fibroin fragments, clusters formed by assemblies or aggregates of silk fibroin particles or smaller silk fibroin fragments, and any combination thereof. In some embodiments, the silk fibroin aggregates can include silk fibroin particles or clusters formed by a process including, but not limited to, precipitation, gelation, and/or clumping. In some embodiments, the silk fibroin aggregates can be silk fibroin particles or clusters having a size of about 1 mm or greater. In one embodiment, the silk fibroin particles or clusters can be formed by assemblies or aggregates of silk fibroin particles present in a solution.

Low molecular weight products:

In some embodiments, the low molecular weight silk fibroin compositions provided herein can form solid silk fibroin articles. Examples of such solid silk fibroin articles can include, but are not limited to, films, sheets, gels or hydrogels, meshes, mats, non-woven mats, fabrics, scaffolds, tubes, blocks, fibers, particles, powders, three-dimensional structures, implants, foams, needles, lyophilized articles, and any combination thereof. Methods for forming various forms of solid silk fibroin articles are known in the art, and some exemplary methods are described herein below.

In some embodiments, the low molecular weight silk fibroin article can be storage stable for at least one month. The term "storage stability" as used herein refers to a material property of silk fibroin particles that remains substantially the same upon storage for a period of time. The material properties of the silk fibroin article can include, but are not limited to, hardness, porosity, solubility, particle size, dryness, and any combination thereof.

In some embodiments, the low molecular weight silk fibroin article can be redissolved in water to form a silk fibroin solution that is substantially free of silk fibroin aggregates. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more of the low molecular weight silk fibroin article can be redissolved in water to form a silk fibroin solution that is substantially free of silk fibroin aggregates.

In some embodiments, heating and/or addition of salt is not required to dissolve the low molecular weight silk fibroin article made using one or more embodiments of the low molecular weight silk fibroin composition described herein. Accordingly, in some embodiments, the silk fibroin article can be re-dissolved in an aqueous solution at room temperature to form a silk fibroin solution. In some embodiments, the silk fibroin article can be completely dissolved in water (e.g., deionized water) at room temperature if the concentration of dissolved silk fibroin is less than saturation. In some embodiments, the saturation point of the low molecular weight silk fibroin composition in an aqueous solution (e.g., water) can be at least 30% w/v, at least 40% w/v, at least 50% w/v, at least 60% w/v, at least 70% w/v, at least 80% w/v or more.

In some embodiments, the saturation point of the low molecular weight silk fibroin composition in an aqueous solution (e.g., water) can range from about 30% w/v to about 80% w/v, from about 40% w/v to about 70% w/v, from about 50% w/v to about 60% w/v. Accordingly, in some embodiments, the low molecular weight silk fibroin article can have an aqueous solubility of at least about 10 mg/mL or more (e.g., at least about 20 mg/mL, at least about 30 mg/mL, at least about 40 mg/mL, at least bout 50 mg/mL, at least about 60 mg/mL, at least about 70 mg/mL, at least about 80 mg/mL, at least about 90 mg/mL, at least about 100 mg/mL, at least about 200 mg/mL, at least about 400 mg/mL, at least about 600 mg/mL, at least about 800 mg/mL, at least about 1000 mg/mL or more).

In some embodiments, the low molecular weight silk fibroin article can have a dissolution rate of at least 0.01 mg/s to about 100 mg/s, or from about 0.1 mg/s to about 90 mg/s, or from about 1 mg/s to about 80 mg/s. In some embodiments, the low molecular weight silk fibroin article can dissolve in an aqueous solution (e.g., water, such as deionized water) at room temperature at a rate of from about 0.01 mg/s to about 100 mg/s, or from about 0.1 mg/s to about 90 mg/s, or from about 1 mg/s to about 80 mg/s.

In some embodiments, the low molecular weight silk fibroin article can be used for on-demand or field diagnostic purposes. For example, a user (e.g., a subject or a healthcare provider) can apply or self-administer the silk fibroin article at any location (e.g., a residence, a rural area, a battlefield). For diagnostic purposes, in some embodiments, the user can send the silk fibroin article to a hospital, clinic, or any analytical facility. Alternatively or additionally, in some embodiments, the low molecular weight fibroin article can be used to generate a stored sample (e.g., a stored blood sample), and in some embodiments, the stored sample can be stable for at least a certain period of time (e.g., days, weeks, months, years, etc.) and/or can be re-tested or sampled over time (e.g., to allow comparison of examples with later obtained samples, etc.).

Without wishing to be bound by theory, in some embodiments, the solubility (i.e., the ability of the solid form of the low molecular weight silk fibroin composition to dissolve or reconstitute into a solution or liquid form) may be determined by the length of time and/or temperature for refining the cocoons. For example, in some embodiments, the soluble silk fibroin article can be formed from a low molecular weight silk fibroin solution, which is obtained by refining the cocoons at or near ambient boiling temperature for at least 60 minutes or more, such as at least 90 minutes or more. In other embodiments, the soluble silk fibroin article can be formed from a low molecular weight silk fibroin solution, which is obtained by refining the cocoons at a lower temperature, such as about 60-90° C., for at least 90 minutes or more, such as at least 120 minutes or more.

In some embodiments, the solid form of the low molecular weight silk fibroin compositions described herein is highly soluble. Thus, in one aspect, the present invention provides a highly soluble solid of a solid silk fibroin composition comprising low molecular weight silk fibroin fragments. Compared to conventional silk fibroin compositions previously described, the highly soluble form of the solid silk fibroin compositions described herein is characterized by its high water solubility, high dissolution rate, as well as biocompatibility with a wide range of agents (such as, but not limited to, biological agents). From a materials science perspective, the low molecular weight silk fibroin compositions described herein provide greater flexibility, compatibility, and ease of processing and denaturation due to the ability to better control liquid-solid or solid-liquid transitions. This is at least in part based on the fact that the solubility of the silk fibroin can be finely controlled when the composition is comprised predominantly of smaller fragments of silk fibroin (i.e., low molecular weight silk fibroin fragments). This added flexibility allows the silk-based material to be utilized in ways that are not possible with conventional silk materials comprising or made of higher molecular weight silk fibroin. Accordingly, the unique material properties of the lower molecular weight silk fibroin materials include, but are not limited to, solid form silk fibroin compositions that are readily soluble in aqueous solutions; and liquid form silk fibroin compositions (e.g., silk fibroin solutions) that resist undesirable self-assembly or gelation while providing a desired stabilizing effect for agents included or associated therewith.

Accordingly, in some embodiments, the low molecular weight silk fibroin compositions can be specifically formulated to behave in a completely soluble form, which can be readily and rapidly reconstituted in water (e.g., distilled or deionized water) or any other water-based solution suitable for a particular application, such as a buffer. In this embodiment, since the low molecular weight silk fibroin compositions in solid form described herein are readily soluble in aqueous solutions, one or more agents introduced therein can be readily recovered from the soluble form of the solid low molecular weight silk fibroin composition. For example, a biological agent, such as a biological sample, can be "stored" together with the low molecular weight silk fibroin composition in solid form, which can then be released into an aqueous solution, thereby achieving nearly complete recovery of the agent. Once released, such agents can be readily analyzed (e.g., detected, measured, assayed, identified, isolated, etc.) by any suitable means, such as an analytical tool. Alternatively or additionally, in some embodiments, the included agent (e.g., a biological agent) can be analyzed without being released from the silk fibroin composition.

Stabilizing Composition

Since activators and/or biological samples are typically unstable and sensitive to changes in ambient conditions, such as temperature, humidity and/or light, stabilization and subsequent recovery of the desired agent to be stabilized (e.g., activator and/or biological sample) is a critical feature of many applications. In many of the previously described methods, even if an activator or biological sample is identified as useful for a given reaction, its use is often hampered by lack of long-term storability and/or low recovery under process conditions. Various embodiments of the present invention described herein, as further described below, address this deficiency present in the prior art.

For example, a technique capable of protecting a stabilized sample against adverse temperature profiles can expand the scope of a centralized test system on a global scale. To this end, the inventors have discovered that certain silk-based materials, among others, can protect an activator (e.g., a biological sample or a component thereof, a therapeutic and/or diagnostic reagent, etc.) from degradation of its structural and/or functional properties. The term "activator" is used herein to refer to a biological sample (e.g., a sample of tissue or a body fluid, such as blood) or a component thereof, and/or a biologically active entity or compound, and/or a structurally or functionally unstable entity.

In some embodiments, stabilization of the bioactivity of the activator/biological sample is very important. In other embodiments, stabilizing activity or bioactivity is not always required. For example, maintenance of bioactivity is often not a requirement for analyzing and quantifying a stabilized biological sample.

In some specific embodiments, the present disclosure demonstrates that, inter alia, silk-based materials can protect human whole blood and blood components, such as RNA, over long periods of time and in the context of adverse environmental conditions. For example, the present disclosure demonstrates that whole blood and/or donor-compatible plasma can be stabilized in respective plasma proteins recovered by a simple mixing protocol with a silk-based material, such as a film, and water, by template or air drying.

The present disclosure also specifically demonstrates various specific advantageous characteristics of low molecular weight silk compositions for stabilizing applications of the included agent, as described herein. For example, among other things, the present disclosure demonstrates that low molecular weight silk compositions tend to gel less rapidly than higher molecular weight silk compositions. Among other things, this delayed gelation property may contribute to extended shelf life in some embodiments. Additionally, the present disclosure demonstrates that low molecular weight silk compositions protect against sample loss even upon autoclaving. In some embodiments, the present invention provides autoclaved low molecular weight silk compositions, including, for example, the included agent. In some embodiments, the present invention provides systems for reconstituting low molecular weight silk compositions (e.g., solid form low molecular weight silk compositions), including such autoclaved compositions, wherein the compositions are characterized by high recovery of the included agent. In certain embodiments, such compositions and/or systems are suitable for large scale manufacturing and/or processing (e.g., shipping) of the compositions.

The stabilizing effects of silk fibroin compositions as demonstrated herein have broad implications, including, for example, for the maintenance, storage, and/or transport of samples obtained in the field (e.g., blood samples). Therefore, in many embodiments, the provided technology is particularly useful in environments where the sample (e.g., collected blood) is obtained from a limited resource and/or where downstream analysis is beyond the reach of point-of-care diagnostic technologies.

This disclosure specifically demonstrates the breadth of clinically relevant analytical tools that can be used to measure the vast majority of blood analytes (or blood components) from a variety of donors once recovered from silk-based materials. The studies reported herein demonstrate that, by using the compositions and methods of the various embodiments described herein, the recovered analyte levels (or recovered component levels) are consistent with measurements performed on donor-matched fresh or frozen plasma, the format currently used as the gold standard for clinical post-processing. In some embodiments, the silk-stabilized analyte levels (or silk-stabilized component levels) are higher than their frozen counterparts, indicating that silk-based materials may outperform the current gold standard for certain applications. The inventors have also demonstrated that different techniques, such as lyophilization, can be used to stabilize biological samples, such as RNA, in the presence of the same silk preparation for subsequent recovery using a simple aqueous reconstitution method. The recovered RNA was usable at levels consistent with frozen controls and capable of transfecting model cell lines.

Silk-based materials in various forms can protect biological samples from widely-varying temperature profiles and from mechanical disturbances encountered during transport. Silk-based materials can be specifically formulated to function as fully dissolved systems upon reconstitution in water (e.g., pure water or a buffer suitable for sample interaction), thereby providing complete recovery of trapped components or biomarkers to be analyzed by a variety of clinically-relevant analytical tools. Prior to the present disclosure, no technology existed in the art that combined these unique attributes: i) ease of sample procurement, ii) impressive thermal/mechanical stability profile, and iii) simplicity of reconstitution/recovery. However, as demonstrated herein, the present disclosure provides compositions and methods which provide, among other things, i) ease of sample procurement, ii) impressive thermal/mechanical stability profile, and iii) simplicity of reconstitution/recovery.

Since silk fibroin is highly resistant to enzymatic/thermal/UV degradation, it is believed that trapped biological samples could be stored in centralized facilities for long-term storage (i.e., months to years) without the need for refrigeration/freezing, to collect longitudinal data sets across entire populations or to improve personalized medicine.

Thus, in one aspect, the present disclosure provides uses of silk solutions (e.g., adjusted to produce silk-based materials having desirable solubility characteristics) and silk-based materials made from these solutions for trapping and stabilizing biological samples, such as whole blood and blood components. The silk purification and trapping scheme provides the ability to completely reconstitute trapped blood components (e.g., cells, and/or nucleic acids such as circulating DNA or RNA). In one embodiment, drawn blood corresponding to a volume obtained by a finger prick can be mixed with a silk solution, such as those described in the Examples section herein, and the at least partially dried silk-based material can then be readily obtained, including the stabilized blood components.

The silk trapping/stabilization platform described herein essentially provides that, for example, a liquid silk fibroin composition can be readily subjected to conventional liquid assays for a biological sample within the composition and subjected to the same assessment and quality control (QC) procedures used for fresh whole blood or frozen samples. The solution composition can be at least partially dried or formed into a silk fibroin article. The silk fibroin within the dried composition or article can be degraded to provide a solution that is amenable to conventional liquid assays for detection of a biological sample or component.

Conversely, because DBS analytes require some level of chemical interaction with the paper matrix when dried to become protected, partial sample retention within the matrix and/or analyte degradation is expected when using standard DBS recovery procedures. As a result, this imposes a unique assessment protocol and QC matrix on DBS post-processing to monitor for loss and inhomogeneity between extractions. Second, the inhomogeneity of spotting across and through the thickness of the paper matrix (from inter-user variability and inter-sample variability of hematocrit) can make it difficult to define sample volume and subsequently appropriately partition paper-based DBS samples. This is particularly problematic for patient populations that vary greatly in age or have abnormal hematocrit levels (e.g., renal impairment, oncology patients). Conversely, the silk-based materials described herein (e.g., low molecular weight silk fibroin compositions) can provide complete recovery when desired (e.g., the entire volume, e.g., an entire 50 μL volume, can be reconstituted), or aliquots can be readily prepared by weight because the silk/blood complex is uniformly mixed or dried.

Accordingly, various aspects of the embodiments described herein also relate to silk-based materials for stabilizing one or more components of a mixed or trapped biological sample, enabling subsequent detection of the components, and methods for making and using the same.

In one aspect, provided herein is a silk-based material comprising silk fibroin (e.g., a low molecular weight silk fibroin composition) and a biological sample/active agent, wherein one or more properties of one or more components of the biological sample are stabilized over a period of time and/or one or more components of the biological sample/active agent are detectable after a period of time.

The properties of one or more active agents (e.g., biological samples or components) stabilized within the silk-based material can be physical or structural properties, chemical properties, and/or biological properties. In some embodiments, the one or more properties can include activity or bioactivity of the agent, structural integrity, structural conformation, and/or amount. Depending on the different applications (e.g., diagnostic versus therapeutic purposes), the silk fibroin and/or silk-silk-based material can be treated to stabilize one or more properties of one or more components of the biological sample.

In some embodiments, stabilization activity or bioactivity of a component of a biological sample is not always required. For example, for diagnostic purposes, the activity of the component to be detected may not be as important as the integrity and/or amount of the stabilized component in the biological sample. In these embodiments, the component to be detected may be inactive, so long as the component remains intact and/or the amount of the component is maintained. The terms "activity" and "bioactivity," as used interchangeably herein, refer to one or more structural and/or functional properties of a component, such as, for example, the ability to interact with and/or exert an effect on its biological target. For example, bioactivity may include, without limitation, the exertion of a stimulatory, inhibitory, modulatory, toxic, or lethal response in a biological target. The biological target may be a molecule or a cell. For example, bioactivity can refer to the ability of a component (e.g., a protein or nucleic acid molecule) to modulate the effect/activity of a cell or enzyme, block a receptor, stimulate a receptor, modulate the expression level of one or more genes, modulate cell proliferation, modulate cell division, modulate cell morphology, infect or transfect a cell, or any combination thereof. In some cases, bioactivity can refer to the ability of a component to produce a toxic effect in a cell.

In some embodiments, it may be desirable to further stabilize the activity of a component of the biological sample. By way of example only, a protein present in the biological sample may be in an active or inactive state. In such embodiments, the silk-based material may stabilize a protein present in the biological sample in its active or native form, such as a phosphorylated protein, or a glycosylated protein. Such embodiments may be useful where the activity of the component plays a role in the diagnosis of a disease or disorder.

According to various embodiments described herein, one or more properties of one or more components of a biological sample can be stabilized within a silk-based material for a period of time. The terms "stabilize," "stabilizing," or "stabilization," as used herein, refer to one or more properties (e.g., activity, integrity, and/or amount) of a component of a biological sample being partially or fully maintained or retained within a silk-based material for a period of time. With respect to stabilization of activity, in some embodiments, at least about 30% or more (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of the component originally present in the biological sample can retain at least partial or full activity for a period of time. In other words, the component can retain at least about 30% (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of its original activity for a period of time. With respect to stabilization of integrity, at least about 30% (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of the component originally present in the biological sample can retain integrity for a period of time. In connection with stabilization of a quantity, at least about 30% (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of a component originally present in the biological sample can be retained for a period of time, regardless of whether the component is active or inactive, or intact or non-intact. The terms "original" or "original" as used herein when used in reference to the original presence or original activity of a component refer to the level of the component as measured immediately after the biological sample is obtained from the subject, or alternatively, immediately before or immediately after the biological sample is mixed or trapped within the silk-based material. In some embodiments, the original presence or original activity of a component refers to the level of the component in a control, e.g., a control that has been stabilized by a non-silk approach, e.g., a frozen control.

One or more properties of the active agent/biological sample/component thereof can be stabilized within the silk-based material for a period of time, e.g., until the component can be extracted or recovered for detection. In some embodiments, the one or more properties can be stabilized for at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours or more. In some embodiments, the one or more properties can be stabilized for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days or more. In some embodiments, the one or more properties can be stabilized for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 6 years or more.

As further discussed below, the silk-based material described herein can be in any form. For example, the silk-based material can be in the form of a solution, a film, a fiber, a particle, a gel, a hydrogel, a foam, a sponge, a mat, a mesh, a fabric, a powder, a coating layer, a lyophilized form thereof, or any combination thereof. In some embodiments, the silk-based material can be a thin film. In some embodiments, the silk-based material can be lyophilized.

One or more components of a biological sample may be detected without isolating them from the silk-based material, but measurable or detectable levels of the component(s) may alternatively be extracted or recovered from the silk-based material for subsequent processing, analysis, and/or characterization. The term "measurable or detectable level" as used herein refers to a lower limit of detection, which generally depends on the sensitivity of the detection and/or the characterization method or assay selected for the particular component. For example, when the component to be detected is a nucleic acid molecule (e.g., RNA or DNA) and an amplification-based detection method such as, but not limited to, polymerase chain reaction (PCR) is selected to detect the component, about 0.0001% of the nucleic acid molecules (e.g., one copy of an RNA molecule) can be extracted or recovered from the silk-based material for amplification and detection.

In some embodiments, the silk-based material can be degradable such that measurable or detectable levels of the component(s) are accessible or usable for subsequent processing, analysis, and/or characterization. As used herein, the term "degradable" generally refers to the solubility, dissolution, or degradation of a silk-based material in a fluid. A degradable silk-based material can be partially or completely degraded or dissolved in a fluid over a period of time. For example, at least about 5% or more of the silk-based material, including, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or up to 100% of the silk-based material, can be degraded or dissolved in a fluid (e.g., an aqueous fluid) over a period of time ranging from seconds, minutes, hours, to days. In some embodiments, the silk-based material can be partially or completely (e.g., at least about 5% or more of the silk-based material) degraded or dissolved in a fluid (e.g., an aqueous fluid) within at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, at least about 40 seconds, at least about 50 seconds, at least about 60 seconds or more. In some embodiments, the silk-based material can be partially or completely (e.g., at least about 5% or more of the silk-based material) degraded or dissolved in a fluid (e.g., an aqueous fluid) within at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 30 minutes, at least about 1 hour or more. In other words, a degradable silk-based material is a silk-based material that can degrade or dissolve a sufficient portion of itself in a fluid (e.g., an aqueous fluid) to recover or extract one or more active agents/samples/components present therein.

The degree of degradability or solubility of the silk-based material may vary depending on several factors, including but not limited to, the composition of the silk-based material (e.g., the ratio of biological sample to silk fibroin, the type of biological sample mixed or trapped with the silk fibroin (e.g., but not limited to, urine, blood, and/or earwax), and/or the type of buffer/excipient used to aid in stabilizing the biological sample and/or one or more of its components, the method of purifying the silk (e.g., refining conditions such as boiling time), the method of forming the silk-based material as described herein, the method of sterilization, and any combination thereof.

In some embodiments, the decomposition or solubility of the silk-based material can be controlled by the molecular weight/chain length of the silk. In general, silk-based materials comprising silk fibroin having lower molecular weight/chain length can have higher solubility in aqueous solvents than silk fibroin having higher molecular weight/chain length. In one embodiment, the molecular weight/chain length of the silk fibroin can be controlled by refining conditions. For example, the silkworm cocoons can be boiled in a salt solution (e.g., Na ₂ CO ₃ ) for a predefined period of time, such as in the range of about 1 minute to about 3 hours, about 5 minutes to about 2 hours, about 10 minutes to about 1.5 hours, or about 15 minutes to about 1 hour. In some embodiments, the silkworm cocoons can be boiled in a salt solution (e.g., Na2CO3) for at least about 1 minute, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, or more. Without wishing to be bound by theory, it is believed that longer boiling times will yield silk fibroin of lower molecular weight/chain length. With respect to the effect of scouring on silk material properties, including molecular weight, viscosity, diffusivity, and degradation behavior, see, e.g., Pritchard EM et al. “Effect of Silk Protein Processing on Drug Delivery from Silk Films” Macromol Biosci. Epub 2013 Jan 24. Depending on the type of biological sample to be stabilized and its component(s), e.g., as described in the Examples, an optimal scouring time can be determined for the desired solubility of the silk-based material and thus recovery of the components. In some embodiments, the silk-based stabilizing composition described herein is a low molecular weight silk composition.

Indeed, the inventors have found that low molecular weight silk fibroin compositions in various forms provide particularly advantageous properties for protecting and/or stabilizing agents (e.g., biological samples) associated with or contained therein. For example, the low molecular weight silk compositions described herein can act as "molecular stabilizers" to protect biological samples from widely varying environmental factors, such as fluctuating temperature profiles, and/or mechanical disturbances encountered during transport, storage, and/or handling, for example. The inventors have also found that compositions comprising low molecular weight silk fibroin described herein can be fully reconstituted in water. In contrast, high molecular weight silk fibroin does not fully reconstitute in water under similar conditions. High molecular weight silk fibroin forms aggregates when reconstituted in water under similar conditions.

To date, no technology has been described in the art that combines these unique properties, namely i) ease of sample procurement, ii) impressive thermal/mechanical stability profile, iii) simplicity of reconstitution/recovery and yield. However, as demonstrated herein, the present disclosure provides compositions and methods which provide i) ease of sample procurement, ii) impressive thermal/mechanical stability profile, iii) simplicity of reconstitution/recovery and yield. Without wishing to be bound by theory, it is believed that silk fibroin is highly resistant to enzymatic/thermal/UV degradation, and that agents (e.g., active agents and/or biological samples) trapped within the silk fibroin compositions can be stored for long-term storage (i.e., for periods of months to years) in a centralized facility without the need for refrigeration/freezing, and can then be readily recovered whenever the agents are ready for use and/or analysis. Thus, the silk fibroin compositions provided herein, and particularly low molecular weight silk fibroin compositions, may enable the collection of longitudinal data sets for entire populations or the improvement of personalized medicine.

The inventors have discovered that incorporating a biological sample into one or more embodiments of the low molecular weight silk fibroin compositions described herein, in particular, stabilizes a detectable moiety present in the biological sample for a period of time under certain conditions, which may consequently enable higher recoveries of the detectable moiety compared to recovery of the detectable moiety from conventional silk fibroin-based materials, for example, for subsequent analysis and/or characterization of the component.

In some embodiments, the present disclosure provides uses for trapping, stabilizing, and subsequently recovering a sample of a low molecular weight silk fibroin composition described herein (e.g., tailored to produce a variety of soluble or solid forms of low molecular weight silk fibroin-based materials, such as solutions, films, particles or powders, scaffolds, meshes, and/or fibers, having desirable properties such as improved solubility (compared to conventional silk fibroin-based materials), improved recovery of additives (compared to conventional silk fibroin-based materials), etc.). In some embodiments, the sample is a biological sample.

The amount of silk fibroin relative to the active agent/biological sample in the silk fibroin compositions described herein can be adjusted for a variety of factors including, but not limited to, for example, the volume and/or type of biological sample, the concentration of silk fibroin, the solubility of the resulting silk-based material, the abundance of the target component to be stabilized and/or detected, the recovery efficiency of the component present in the biological sample, and the detection sensitivity of the detection/characterization method selected for a particular component.

In some embodiments, the ratio (e.g., mass ratio, volume ratio, or molar ratio) of silk fibroin to active agent/sample/component is in the range of from about 1:10000 to about 10000:1, or from about 1:1000 to about 1000:1. In some embodiments, the ratio (e.g., mass ratio, volume ratio, or molar ratio) is from about 1:1 to about 1000:1. In some embodiments, the ratio is about 1:1000 to about 1000:1, about 1:500 to about 500:1, about 1:250 to about 250:1, about 1:125 to about 125:1, about 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to about 25:1, about 1:10 to about 10:1, about 1:5 to about 5:1, about 1:3 to about 3:1, or about 1:1. By way of example only, in some embodiments, the volume ratio of silk fibroin to blood sample can be in the range of about 1:1 to about 1000:1, or about 1:1 to about 100:1, or about 1:1: to about 50:1, or about 1:1 to about 25:1.

The ratio of silk fibroin matrix to active agent/sample/ingredient can vary depending on several factors including the choice of active agent, storage conditions and duration, concentration of silk fibroin matrix, and morphology of the silk matrix. One of ordinary skill in the art can determine the appropriate ratio of silk fibroin matrix to active agent by, for example, measuring the bioactivity of the active agent maintained at various ratios described herein for a predefined period of time under defined conditions, for example, at a temperature above 0° C. Methods for measuring the bioactivity of various active agents, such as enzymes, vaccines, proteins, antibodies, and nucleic acids, described herein are known in the art. For example, the stability or bioactivity of a given active agent in silk fibroin can be determined based on a combination of time and temperature. For example, a stabilization study can be performed for 6 months. Activity assays can be performed, for example, after 2 weeks, 4 weeks, and then monthly. Samples can be prepared to provide N=3 for each time point. The range of temperature storage conditions to be evaluated includes 4°C (refrigerated), up to 25°C (room temperature), 37°C (body temperature), 45°C and/or 50°C. Alternatively or additionally, activity can be assayed after 1, 2, 3 or more freeze-thaw cycles. These variables can be fully combined to fully characterize an optimal formulation for long-term stability of the active agent(s). In some embodiments, the results of the silk-related active agent stability can be compared to, for example, a lyophilized active agent formulation under the same storage conditions, for the purpose of improving stability at the manufacturer-recommended storage conditions of the lyophilized active agent formulation (e.g., 4°C).

The stabilized silk fibroin compositions described herein can include any amount of silk fibroin, so long as the amount is sufficient to stabilize one or more properties of one or more components of a biological sample for a period of time and to allow detection of the active agent/sample/component after the period of time. In some embodiments, the amount of silk fibroin in the composition will vary depending on, for example, the type and/or volume size of the active agent/sample/component described herein, the form of the silk-based material (e.g., film versus foam), the method of making any silk-based material to be formed (e.g., air-dried versus lyophilized), and/or the concentration of silk fibroin used to form the initial silk fibroin composition.

For example, the silk-based stabilizing material can be prepared from an agent-containing silk solution comprising about 0.25% to about 50% (w/v) silk fibroin, or about 0.5% to about 30% (w/v), or about 0.5% to about 15% (w/v), or about 0.5% to about 10% (w/v). In some embodiments, the silk-based material can be a silk solution comprising a biological sample. For example, in one embodiment, a silk-based film can be prepared from a silk solution comprising about 1% to about 15% (w/v) silk fibroin and an active agent or biological sample/component. In another embodiment, a silk-based foam can be prepared from a silk solution comprising about 0.1% to about 5% (w/v) silk fibroin and an active agent/biological sample/component.

In some embodiments, the silk-based material can be a solid-state silk-based material formed from a silk solution comprising a biological sample.

In some embodiments, the agent/sample/component can be stabilized in a silk composition (e.g., in a low molecular weight base composition) at a range of storage temperatures for a period of time as described herein and can allow for recovery and/or detection of one or more components of the biological sample for analysis. Unlike the conventional sub-zero storage temperatures required for biological samples (e.g., in a -80° C. freezer or in liquid nitrogen), the silk-based materials described herein allow for biological samples to be stabilized therein at storage temperatures above 0° C. or greater. In some embodiments, the storage temperature can be in the range of about 0° C. to about room temperature. In some embodiments, the storage temperature can be at least about 40° C. or greater than 40° C. In some embodiments, the storage temperature can be at least about 45° C. or greater than 45° C.

In some embodiments, the agent/sample/component can be stabilized within the silk fibroin composition described herein under light exposure for a period of time as described herein and can allow for recovery and/or detection of one or more components of the biological sample for analysis. For example, in some embodiments, the agent/sample/component present within the composition can be exposed to light, e.g., light of different wavelengths and/or from different sources. In some embodiments, the agent/sample/component present within the silk-based material can be exposed to UV or infrared irradiation. In some embodiments, the agent/sample/component present within the silk-based material can be exposed to visible light.

In some embodiments, a biological sample can be stabilized within a silk fibroin composition provided herein at a range of relative humidity for a period of time and can enable recovery and/or detection of one or more components of the biological sample for analysis. In some embodiments, the relative humidity can be at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% or more.

The term "relative humidity" as used herein is a measure of the amount of water vapor in a mixture of air and water vapor. It is generally defined as the partial pressure of water vapor in an air-water mixture, and is given as a percentage of the saturation vapor pressure under those conditions.

In some embodiments, the stabilized silk fibroin composition described can be lyophilized, for example, to reduce the residual moisture during storage. In some embodiments, the residual moisture is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%.

In some embodiments, the compositions described herein can be maintained or exposed to any air pressure. In some embodiments, the compositions described herein can be maintained or exposed to about atmospheric pressure, or higher, for example, about 1 atm, about 2 atm, about 3 atm, about 4 atm, about 5 atm, about 6 atm, about 7 atm, about 8 atm, about 9 atm, or about 10 atm. In some embodiments, the compositions described herein can be maintained or exposed to a vacuum.

Although not required, in some embodiments where the silk fibroin composition (e.g., a silk-based solution) is stored at sub-zero temperatures, the agent/sample/component can be stabilized within the composition upon at least one or more freeze-thaw cycles (e.g., a frozen silk-based solution, a thawed silk-based solution) and can enable recovery and/or detection of one or more components of the biological sample for analysis. The term "freeze-thaw cycle" is used herein to describe a series of alternating freezes and thaws and also to include a series of alternating frozen (solid) and fluid states. For example, a freeze-thaw cycle involves a change of state between a frozen (solid) state and a fluid state. The time interval between the freeze and thaw, or the frozen and fluid state, can be any period of time, such as hours, days, weeks, or months. For example, once the silk-based solution is frozen or in a frozen state, it can be stored continuously at sub-zero temperatures, for example, at about -20° C. to -80° C., until it needs to be thawed for later use. Freezing of the composition can be accomplished rapidly, for example, in liquid nitrogen, or gradually, for example, at freezing temperatures, for example, at about -20° C. to -80° C. Thawing of the frozen composition can be accomplished rapidly, for example, at room temperature, or gradually, for example, on ice, at any temperature above 0° C. Typically, components of the biological sample within the non-silk fibroin matrix, for example, proteins and/or nucleic acids, can lose their activity or integrity during one or more freeze-thaw cycles. Distributing the biological sample within the silk fibroin matrix, as described herein, can enhance the stability of one or more components thereof and thus retain their activity or integrity during one or more freeze-thaw cycles.

In one embodiment, the agent/sample/component can be stabilized within the silk fibroin composition provided herein under two or more of the conditions described above for a period of time as described herein, allowing for recovery and/or detection of one or more components of the agent/sample/component for analysis.

Another aspect provided herein relates to methods and compositions for maintaining or stabilizing the bioactivity of an active agent. The methods comprise maintaining a composition for a period of time, wherein the composition comprises a silk fibroin matrix and one or more active agents distributed, mixed, or embedded therein, wherein when the composition is exposed to specified conditions that inhibit or reduce the bioactivity of the active agent, the one or more active agents retain or stabilize at least about 30% of their original bioactivity. Such conditions can include, but are not limited to, state-change cycles, temperature, air pressure, humidity, and exposure to light.

The term "state-change cycle" as used herein refers to a change in the state of a substance, including but not limited to a solid state to a fluid state, or a fluid state to a solid state. A fluid state can include, but is not limited to, a liquid, a gas, a slurry, a fluid paste, a plasma, and any combination thereof. A solid state refers to a state that is not fluid, and can also include a semi-solid, such as a gel. A composition described herein can remain in a characteristic state for any period of time, such as seconds, minutes, hours, weeks, months, or years, before changing to another state. A state-change cycle can be due to one or more changes in environmental conditions described herein, such as changes in temperature, changes in ambient pressure, light conditions, humidity, or any combination thereof.

In one embodiment, the state-change cycle represents a freeze-thaw cycle. In such embodiments, the compositions described herein can be subjected to one or more freeze-thaw cycles, two or more freeze-thaw cycles, three or more freeze-thaw cycles, four or more freeze-thaw cycles, five or more freeze-thaw cycles, six or more freeze-thaw cycles, seven or more freeze-thaw cycles, eight or more freeze-thaw cycles, nine or more freeze-thaw cycles, ten or more freeze-thaw cycles, or more, when stored or transported. The term "freeze-thaw cycle" is used herein to describe a series of alternating freezes and thaws, and also includes a series of alternating frozen (solid) and fluid states. For example, a freeze-thaw cycle involves a state change between a frozen (solid) state and a fluid state. The time interval between freezing and thawing, or between freezing and fluid states, can be any period of time, such as hours, days, weeks, or months. For example, once the active composition is frozen or in a frozen state, it can be continuously stored in a frozen state at sub-zero temperatures, for example, at about -20° C. to -80° C., until it needs to be thawed for further use. Freezing of the composition can be accomplished rapidly, for example, in liquid nitrogen, or gradually, for example, at freezing temperatures, for example, at about -20° C. to -80° C. Thawing of the frozen composition can be accomplished rapidly, for example, at room temperature, or gradually, for example, on ice, at any temperature above 0° C. Typically, the active agent within the non-silk fibroin matrix can lose its bioactivity upon one or more freeze-thaw cycles. Distributing the active agent within the silk fibroin matrix, as described herein, can enhance the stability of the active agent and thus allow it to maintain its bioactivity during one or more freeze-thaw cycles.

Embodiments of the various aspects described herein provide stabilized actives, wherein stabilization of the active is achieved by distributing, mixing, or embedding the active within a silk fibroin composition described herein. The silk fibroin can be a silk fibroin solution or a solid-state silk fibroin matrix, e.g., a silk fibroin article. This approach allows the active to retain its bioactivity regardless of the refrigerated distribution and/or environmental conditions under which the active is stored and/or transported. Exemplary environmental conditions include, but are not limited to, temperature, air pressure, humidity, and light exposure. For example, refrigerated distribution is a standard relationship for stabilizing actives in the pharmaceutical industry: maintaining refrigerated distribution ensures that the active is transported and stored according to the manufacturer's recommended temperature range (e.g., 2°C to 8°C or sub-zero temperatures) until use.

In some embodiments, the silk fibroin composition comprising the active agent is in the form of an implant or an implantable drug delivery device. The active agent in such composition can retain at least 30% (including at least about 40%, at least about 60%, at least about 80%, or more) of its original bioactivity or more for a period of time. In some embodiments, the active agent in the silk fibroin composition in the form of an implantable drug device can retain at least about 30% of its original bioactivity or more for at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or at least 1 year or more after implantation.

In some embodiments, one or more active agents encapsulated in the injectable silk fibroin composition can be administered to a subject (e.g., by injection, such as a subcutaneous injection) from a reservoir of the active agent (e.g., a vaccine reservoir) such that the active agent (e.g., a vaccine) can be released from the reservoir continuously or intermittently over an extended period of time, such as for hours, days, weeks, or months. In some embodiments, the active agent (e.g., a vaccine) can be released at a rate such that greater than or equal to about 1% (including at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more) of the encapsulated active agent is released over a period of at least 1 hour, at least 2 hours, at least 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, or more. In some embodiments, the active agent (e.g., a vaccine) can be released at a rate such that greater than or equal to about 10% (including at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more) of the encapsulated active agent is released over a period of 5 days, a period of 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, or more.

In some embodiments, the active agent in the silk fibroin composition retains at least about 30% of its original bioactivity, for example, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or higher of its original bioactivity at about 4° C., at about 25° C., at about 37° C., at about 45° C., or higher, for at least 6 months. In some embodiments, the active agent retains at least about 8% of its original bioactivity at a temperature of at least about 37° C.

Certain aspects described herein are storage-stable compositions comprising a silk fibroin composition (e.g., a low molecular weight silk fibroin composition) and an active agent dispersed, mixed or embedded therein, wherein the active agent retains at least about 30% of its original bioactivity (e.g., at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, etc.) when the composition is exposed to one or more state-change cycles and/or maintained for a period of time under one or more conditions specified herein. In some embodiments, the active agent can be covalently or non-covalently fused or linked to a silk fibroin fragment in the composition. In one embodiment, the state-change cycle is a freeze-thaw cycle. In one embodiment, the period of time for maintaining the active agent is at least about 24 hours. In some embodiments, the specified conditions may be environmental conditions under which the active agent is stored and/or transported. Non-limiting examples of environmental conditions include temperature, air pressure, humidity, and exposure to light. In some embodiments, the compositions described herein may be immunogenic. In some such embodiments, the active agent is an immunogen. In some embodiments, the active agent is a vaccine.

In some aspects, as described herein, the present invention provides methods for stabilizing a biological sample or active agent. Generally, the methods comprise contacting, combining, or mixing the biological sample or active agent with a silk fibroin composition described herein, thereby providing a mixture comprising silk fibroin and a biological sample or active agent. In some embodiments, the methods further comprise forming a silk-based material (e.g., a silk fibroin article) from the mixture. Thus, in some embodiments, the methods for stabilizing a biological sample or active agent comprise providing a low molecular weight silk fibroin composition comprising the biological sample or active agent and forming a silk-based article from the composition. In some embodiments, when the silk fibroin composition comprising the active agent/sample/component is in a solid state, it can be further treated to induce the formation of beta-sheet secondary structures within the silk fibroin.

In some embodiments, the method provided comprises the step of contacting a biological sample or an active agent with a silk fibroin composition in powder form. In some further embodiments thereof, the method further comprises the step of forming a solution from the composition.

In some embodiments, the method comprises contacting a biological sample or active agent with a silk fibroin composition in solution form.

In some embodiments, a solution composition comprising a biological sample or an active agent can be formed into a silk fibroin article. The resulting article can be soluble in an aqueous solution (e.g., water, a buffer solution, or a combination thereof).

The amount of silk fibroin to biological sample in the compositions described herein can be adjusted for several factors including, but not limited to, the volume and/or type of biological sample, the concentration of silk fibroin, the solubility of the resulting silk-based material, the degree of stabilization and/or abundance of the target component to be detected, the recovery efficiency of the component present in the biological sample, and the detection sensitivity of the detection/characterization method selected for a particular component. For example, as previously described, in some embodiments, the ratio (e.g., mass ratio, volume ratio, or molar ratio) of silk fibroin to biological sample can be in the range of from about 1:10000 to about 10000:1, or from about 1:1000 to about 1000:1. In some embodiments, the ratio (e.g., mass ratio, volume ratio, or molar ratio) of silk fibroin to biological sample is from about 1:1 to about 1000:1. By way of example only, in some embodiments, the volume ratio of silk fibroin to blood sample can range from about 1:1 to about 1000:1, or from about 1:1 to about 100:1, or from about 1:1: to about 50:1, or from about 1:1 to about 25:1.

Active Agent/Ingredient

The silk composition (e.g., the low molecular weight silk fibroin composition described herein) may include/contain any of a variety of active and/or destabilizing agents.

Non-limiting examples of biological samples that can be stored, stabilized, analyzed, and/or recovered from the silk fibroin compositions described herein include, but are not limited to: bodily fluid samples such as blood samples, e.g., whole blood samples, plasma samples, and serum samples; urine samples; cerebrospinal fluid samples, saliva samples, and any combination thereof. In some embodiments, such samples can be collected from a patient for medical or clinical purposes. In some embodiments, such samples can be collected for forensic purposes.

In some embodiments, the unique properties of the provided silk fibroin compositions (e.g., low molecular weight silk compositions) described herein, for example in the context of agent incorporation, stabilization and recovery, allow for complete reconstitution of various trapped blood components (e.g., cells, circulating factors such as hormones, growth factors, cytokines, antibodies and other proteins, nucleic acids such as DNA or RNA, etc.). For example, drawn blood corresponding to a volume obtained by a finger prick can be mixed with a silk fibroin solution to form an at least partially dried silk-based material, which can then be readily obtained containing the stabilized blood components and stored until ready for use and/or analysis.

In addition to blood samples, other biological samples used for diagnostic purposes, including but not limited to, urine, blood, feces, earwax, nucleic acids (e.g., DNA/RNA, modified nucleic acids), antibodies, whole cells, therapeutic agents, can be contacted with silk fibroin in a similar manner, depending on performance requirements and sample availability. The provided silk fibroin compositions comprising a biological sample can stabilize one or more components of the biological sample for a period of time, which allows for subsequent analysis and/or characterization of the biological sample or components thereof. Accordingly, the various aspects of the embodiments described herein also relate to silk fibroin-based compositions for stabilizing one or more components of a mixed or trapped biological sample, and methods of making and using the same, to enable subsequent detection of the components.

According to various embodiments described herein, the biological sample to be mixed or trapped within the low molecular weight silk fibroin comprises any fluid or specimen (treated or untreated) that is intended to be stabilized for and/or evaluated for the presence of one or more components. The biological sample can be a liquid, a supercritical fluid, a solution, a suspension, a gas, a gel, a slurry, a solid, and combinations thereof.

In some embodiments, the biological sample may comprise an aqueous fluid. As used herein, the term "aqueous fluid" refers to any flowable water-containing substance.

In some embodiments, the biological sample can be obtained from a subject, e.g., a mammalian subject, such as a human subject. Exemplary biological samples obtained from a subject can include, but are not limited to, biological cells, tissues, blood (including whole blood, plasma, cord blood, and serum), breast milk products (e.g., milk), amniotic fluid, sputum, saliva, urine, semen, cerebrospinal fluid, bronchial aspirates, sweat, mucus, liquefied feces, synovial fluid, lymphatic fluid, tears, tracheal aspirates, and fractions thereof. In some embodiments, the biological sample can comprise a homogenate of a tissue specimen (e.g., a biopsy) from the subject. In one embodiment, the biological sample can comprise a suspension obtained by homogenization of a solid sample obtained from a solid organ or a fragment thereof. The biological sample can be obtained from the subject by any means known in the art, which can vary depending on the type of biological sample. By way of example only, a blood sample may be obtained from a subject by finger prick, microneedle prick, venous blood draw, or any known method for collecting blood samples.

In some embodiments, the biological sample can comprise biological cells selected from the group consisting of living and dead cells (including prokaryotic and eukaryotic, mammalian), viruses, bacteria, fungi, yeast, protozoa, microorganisms, and parasites. The biological cells can be normal cells or diseased cells, for example, cancer cells. Mammalian cells include cells from any animal, including, without limitation; primates, humans, and, without limitation; mice, hamsters, rabbits, dogs, cats, birds, domestic animals, such as horses, cows, murines, sheep, dogs, and cats. In some embodiments, the cells can be derived from a human subject. In other embodiments, the cells can be derived from a domesticated animal, for example, a dog or a cat. Exemplary mammalian cells include, but are not limited to, stem cells, cancer cells, progenitor cells, immune cells, blood cells, fetal cells, and any combination thereof. The cells can be derived from a variety of tissue types, including, without limitation; Hematopoietic, neural, mesenchymal, skin, mucosal, stromal, muscle, spleen, reticulothelial, epithelial, endothelial, liver, kidney, gastrointestinal, pulmonary, cardiovascular, T-cell, and fetal. Stem cells, including, without limitation, hematopoietic, neural, stromal, muscle, cardiovascular, hepatic, pulmonary, and gastrointestinal stem cells, embryonic stem (ES) cells, ES-derived cells, induced pluripotent stem cells, and stem cell progenitor cells, are also included. Yeast cells may also be used as cells in some embodiments described herein. In some embodiments, the cells may be ex vivo or cultured cells, e.g., in vitro. For example, in the case of ex vivo cells, the cells may be obtained from a healthy and/or diseased subject.

In some embodiments, the biological sample may include tissue derived from an animal part, such as but not limited to one or more organs (including skin), muscles, beaks, mandibles, feathers, wings, and/or tail.

In some embodiments, the biological sample can include a fluid or specimen obtained from a source including, but not limited to, a laboratory, an animal colony, a crime scene, a cell bank or repository, a blood bank, a tissue bank, a biological specimen repository, a diagnostic testing facility, a clinical setting, and/or any combination thereof.

In some embodiments, the biological sample can include cells from a fluid (e.g., a culture medium) and/or a biological medium. Examples of cells obtained from a fluid (e.g., a culture medium) and/or a biological medium include those obtained from the culture or fermentation of single- or multi-celled organisms, including, for example, prokaryotes (e.g., bacteria) and eukaryotes (e.g., animal cells, plant cells, yeast, fungi), and fractions thereof.

In some embodiments, a biological sample comprises one or more components or a mixture of components. For example, examples of components that can be stabilized for subsequent detection and/or characterization include, but are not limited to, peptides, proteins, antibodies, enzymes, antigens, amino acids, nucleic acids (e.g., DNA, RNA, siRNA, miRNA, non-coding RNA, or any variant of RNA that can be found endogenously in the subject), nucleotides, metabolites, lipids, sugars, glycoproteins, peptidoglycans, microorganisms, cells, and any combination thereof. Using the compositions and/or methods described herein, one or more components, including, for example, at least two components, at least three components, at least four components, at least five components, or more, can be stabilized for a period of time and then detected after a period of time. In some embodiments, the silk-based material can stabilize at least one (e.g., 1, 2, 3, 4, 5, or more) protein(s) in a biological sample (e.g., plasma or serum proteins in a blood sample) that can then be detected. In some embodiments, the silk-based material can stabilize at least one (e.g., 1, 2, 3, 4, 5, or more) cell-free or circulating DNA or RNA in a blood sample. In some embodiments, the silk-based material can stabilize at least one (e.g., 1, 2, 3, 4, 5, or more) diagnostic biomarkers present in a biological sample.

In some embodiments, the silk-based material can stabilize the activity and/or integrity of RNA, and it is contemplated that the silk fibroin compositions and methods described herein can be used to stabilize any nucleic acid, including but not limited to DNA, RNA, and modified DNA or RNA. Some exemplary nucleic acids include peptide plasmid DNA, genomic DNA, mRNA, siRNA, pre-miRNA, miRNA, antisense oligonucleotides, shRNA, activating RNA, decoy oligonucleotides, peptide nucleic acids (PNAs), oligonucleotides, and/or any nucleic acid that can be administered to a subject for therapeutic purposes.

In some embodiments, when incorporating a biological sample with one or more embodiments of the silk fibroin compositions described herein, it may be desirable to stabilize the state of one or more components (e.g., one or more detectable entities) of the biological sample. By way of example only, a protein present in the biological sample may be in an active or inactive state. In some embodiments, an active or native state, e.g., a phosphorylated protein, or a glycosylated protein of a protein of the biological sample, may be stabilized or maintained within the silk fibroin material. These embodiments may be useful, for example, when the activity or state of a component plays a role in the diagnosis of a disease or disorder. For example, a particular state or level of post-translational modification may correlate with a disease state and thus be helpful in diagnosis. In some embodiments, the post-translational modification is: phosphorylation, myristoylation, palmitoylation, isoprenylation or prenylation, farnesylation, geranylgeranylation, glyphiation, glycosylphosphotidylinositol anchor formation, lipoylation, attachment of a flavin moiety, attachment of heme C, phosphopantetheinylation, retinylidene Schiff base formation, diphthamide formation, ethanolamine phosphoglycerol attachment, hippusine formation, acylation, acetylation, formylation, alkylation, methylation, amide bond formation, C-terminal amidation, amino acid addition, arginylation, polyglutamylation, polyglycylation, butyrylation, gamma-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphate ester or phosphoramidate formation, Including but not limited to phosphorylation, adenylation, propionylation, pyroglutamate formation, S-glutathionylation, S-nitrosylation, succinylation, sulfation, selenoylation, glycation, biotinylation, pegylation, ISGylation, SUMOylation, ubiquitination, neddylation, PUPylation, citrullination, deamidation, eliminylation, carbamylation, disulfide cross-linking, proteolytic cleavage, and racemization of proline.

In general, to incorporate a biological sample or agent into the silk fibroin article, the biological sample or agent may be included in the silk fibroin solution used to manufacture the silk fibroin article. Alternatively, or additionally, a pre-formed silk fibroin article may be added to the solution comprising the biological sample or agent, such that the biological sample (or component thereof) or agent is adsorbed into/on the silk fibroin article.

In some embodiments, the biological sample/agent can be distributed homogeneously or non-homogeneously (e.g., in a gradient) in the silk-based material. In some embodiments, the biological sample can be encapsulated or trapped by the silk fibroin in the silk-based material. In some embodiments, the biological sample can be mixed or blended with the silk fibroin within the silk-based material.

After the silk-based material is formed, the material can be treated to, for example, induce the formation of beta-sheet secondary structures within the silk fibroin. Methods for inducing the formation of beta-sheet secondary structures within silk fibroin are described elsewhere herein.

According to various aspects of the embodiments described herein, a silk fibroin composition comprising a biological sample can stabilize one or more components of the biological sample, thereby allowing for subsequent detection and/or analysis of the components. In some embodiments, these silk-based materials comprising a biological sample can be stored and/or transported without requiring refrigeration or freezing, which are conventional methods for currently storing and handling biological samples, to maintain/preserve sample quality for diagnostic assessments of human health. Accordingly, in some embodiments, the silk-based materials and methods described herein can be useful for diagnostic applications. For example, the silk-based materials and methods can be used to maintain and/or preserve the quality of a biological sample during storage and/or transport, or in remote field conditions in some developing countries or where minimal infrastructure to support continuous refrigerated storage does not exist, so that one or more components of the biological sample can be assayed for diagnostic applications. In some embodiments, the silk-based materials and methods described herein can be used to maintain the quality and/or retain biological samples under one or more or any combination of the following conditions, namely: (a) a temperature above 0° C. (e.g., at least about room temperature or higher) during storage and/or transport; (b) exposure to light (e.g., UV, infrared, and/or visible light) during storage and/or transport; and (c) a relative humidity of at least about 10% or higher during storage and/or transport.

Accordingly, another aspect provided herein is directed to a method of using the silk-based material described herein. The method comprises the steps of: (a) providing one or more embodiments of a silk fibroin composition comprising an active agent/sample component (e.g., low molecular weight silk fibroin); and (b) dissolving at least a portion of the composition in water, thereby forming a sample solution comprising silk fibroin and a detectable amount of one or more active agents.

In some embodiments, a subject or patient in need of diagnosis of a disease or disorder can provide a biological sample and contact the biological sample with a silk fibroin composition, which can then be sent to a diagnostic testing laboratory for analysis. In some embodiments, the biological sample can be collected from the subject by a skilled practitioner in a clinical setting, where the biological sample can then be contacted with silk fibroin (e.g., low molecular weight silk fibroin) and sent to a diagnostic testing laboratory for analysis.

In some embodiments, one or more components of a biological sample can be detected and/or analyzed without isolating the components from the biological sample, although in alternative embodiments, the components can be extracted or recovered from at least a portion of the silk fibroin composition prior to detection and/or analysis using any method known in the art. According to some embodiments of the silk-based materials described herein, the composition can be soluble in an aqueous solution (e.g., water, a buffer solution, or a combination thereof). Unlike cellulose-based technologies, such as dried blood spots where blood is blotted onto filter paper and is then difficult to recover, at least a portion of the silk fibroin compositions described herein can be dissolved in an aqueous solution (e.g., water, a buffer solution, or a combination thereof). The resulting silk/biological sample solution can then be subjected to conventional liquid assays, such as ELISA and Luminex™ assays described in the Examples, without further purification. Thus, in some embodiments, the method can further comprise contacting at least a portion of the silk fibroin composition with an aqueous solution (e.g., water, a buffer solution, or a combination thereof) prior to subjecting the one or more components of the biological sample to one or more analyses.

For example, depending on the nature of the target component, various types of analyses can be performed on the target component of the biological sample. Non-limiting examples of analyses can include, but are not limited to, genotyping, nucleic acid sequencing, expression analysis (e.g., protein level, or transcript level), binding affinity, enzymatic activity, transfection efficiency, cell counting, cell identification, cell viability, immunogenicity, infectivity, metabolite profiling, and any combination thereof. In some embodiments, one or more components of the biological sample can be subjected to one or more of genotyping or nucleic acid sequencing analyses, expression analysis (e.g., protein level and/or transcript level), metabolite profiling, or any combination thereof. Various methods for performing these analyses may include, but are not limited to, polymerase chain reaction (PCR), real-time quantitative PCR, microarray, western blot, immunohistochemical analysis, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, nucleic acid sequencing, flow cytometry, gas chromatography, high performance liquid chromatography, nuclear magnetic resonance (NMR) spectroscopy, or any combination thereof. Techniques for nucleic acid sequencing, including but not limited to DNA sequencing, RNA sequencing, de novo sequencing, next generation sequencing including massively parallel signature sequencing (MPSS), polony sequencing, pyrosequencing, Illumina (Solexa) sequencing, solid-state sequencing, ion semiconductor sequencing, DNA nanopore sequencing, heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing), nanopore DNA sequencing, sequencing by hybridization, sequencing using mass spectrometry, microfluidic Sanger sequencing, microscopy-based sequencing techniques, RNA polymerase (RNAP) sequencing, or any combination thereof, are known in the art and can be used to assay a component to elucidate a nucleic acid or gene expression measurement.

In some embodiments, one or more analyses may be performed in a format that can be interfaced with a reading device or system. In some embodiments, one or more analyses may be performed on the system.

In some embodiments, prior to contacting a biological sample comprising an analyte or component with an aqueous solution, the component can be divided or reduced into smaller portions, e.g., a portion thereof can be retained for later analysis and/or analyzed for different target components (e.g., proteins, nucleic acids, and/or metabolites). The division into smaller portions can be by weight or by volume.

The recovery of one or more agents from a silk fibroin composition generally depends, in part, on the solubility of the silk fibroin composition into which the agent is encapsulated, the stability of the agent when present in the silk fibroin composition, and/or the ease of processing of the agent-containing silk fibroin solution for subsequent analysis. As noted herein, the inventors have surprisingly found that, unlike other silk compositions having a large portion of silk fibroin fragments having a molecular weight greater than 200 kDa (e.g., a silk composition prepared from a silk solution obtained by refining silkworm cocoons for less than 30 minutes), the low molecular weight silk fibroin compositions described herein readily dissolve or decompose in water at room temperature without any additives (e.g., salts) or heating to form a silk fibroin solution. For example, in some embodiments, the silk fibroin particles (including powders) and films described herein can readily dissolve or decompose in deionized water at room temperature. Thus, in some embodiments, the resulting silk fibroin solution can be used without any subsequent dialysis that would otherwise normally be required. In some embodiments, the inventors have determined that the silk fibroin solution reconstituted from the low molecular weight silk fibroin compositions described herein can be directly injected into a fluidics-based analytical instrument for subsequent analysis. In addition, the resulting silk fibroin solution can also be used to reconstitute other low molecular weight silk fibroin compositions described herein.

In addition to their unique characteristics of degradability and the ability to reform articles from the resulting silk solution, the low molecular weight silk fibroin compositions described herein can also stabilize at least one agent in a sample for an extended period of time, as compared to the stability of the agent in the absence of the silk fibroin composition, for example, as described in International Application No. PCT/US12/34643 and U.S. Provisional Application Nos. 61/792,161 and 61/830,950. Thus, in some embodiments, the low molecular weight silk fibroin compositions described herein are suitable for any application in which an agent or sample needs to be stabilized for a period of time in order to be recovered or recovered for subsequent use and/or analysis. By way of example only, in some embodiments, an agent or sample encapsulated in one or more embodiments of a low molecular weight silk fibroin article, e.g., a low molecular weight silk fibroin film or powder, can be stored at room temperature without refrigeration for a period of time until ready for use and/or analysis. In these embodiments, at least a portion of the low molecular weight silk fibroin article comprising the agent or sample can be dissolved in water at room temperature to restore the agent or sample to a usable form, while the remainder of the sample can be left for subsequent use and/or other analyses.

In some embodiments, at least a desirable or detectable amount of an agent or sample encapsulated in the low molecular weight silk fibroin compositions described herein can be recovered in a usable form. As used herein, the term "usable form" refers to a form of an agent or sample that is suitable for a particular use. For example, an agent or sample can be recovered from solution for assay analysis. In some embodiments, the term "usable form" can include an agent or sample that has been separated from solution, for example, by any recognized art purification method. Because the low molecular weight silk fibroin composition can stabilize and readily dissolve the agent, a smaller aliquot of the low molecular weight silk fibroin composition can generally be sufficient to provide a desirable or detectable amount of the agent or sample, as compared to recovery of the agent or sample from a non-silk fibroin composition. In other words, the recovery of agent or sample from the low molecular weight silk fibroin compositions described herein, together with fractions containing the same load of agent or sample, can be increased by at least about 10%, including, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the recovery from the non-silk fibroin composition.

In some embodiments, at least about 50% or more of the original load of the agent or sample in the silk fibroin composition (e.g., the low molecular weight composition) can be recovered. In some embodiments, greater than 50% of the original load of the agent or sample in the low molecular weight silk fibroin composition can be recovered, including, for example, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more. In some embodiments, the agent or sample can be recovered by dissolving the low molecular weight silk fibroin article and encapsulating it in room temperature water (e.g., deionized water). In some embodiments, the dissolved silk fibroin article comprising the agent or sample can be subjected to further processing, such as analysis of the agent from the silk fibroin solution, and/or purification of the agent.

Because of the ease of manipulation and/or handling of the low molecular weight silk fibroin compositions described herein, the compositions described herein may be applied during emergency transport and/or for home use, in military field settings, and in clinical settings. By way of example only, to stabilize a blood sample, e.g., for transport from a home or remote field location to a laboratory facility, a home user or military personnel may make a storage-stable low molecular weight silk fibroin composition into a silk fibroin solution, and the blood sample may then be mixed together. The sample solution mixture of components of the blood sample stabilized in the presence of the silk fibroin may then be shipped to the laboratory facility. In some embodiments, the silk fibroin solution comprising the blood sample may be air-dried to form a film that allows the article, e.g., the components of the blood sample, to be stabilized at ambient temperature for a longer period of time, e.g., at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or more. Preserving the activity and/or levels of one or more desirable components in a sample using the low molecular weight silk fibroin compositions and/or methods described herein may aid in the discovery of novel biomarkers for diseases or disorders, increase the sensitivity of diagnostic tests, and/or provide more accurate diagnostic test readouts.

Compositions and methods for stabilizing biological samples using silk fibroin are described in the inventors' U.S. Provisional Application Nos. 61/792,161, filed March 15, 2013 and 61/830,950, filed June 4, 2013, both of which are incorporated herein by reference in their entireties.

Exemplary composition format

Without limitation, the silk fibroin compositions described herein (e.g., low molecular weight silk fibroin compositions and/or silk fibroin compositions comprising biological samples or active agents for stabilization) can be of any shape, shape, or size. For example, the composition can be a solution, a fiber, a film, a sheet, a fiber, a mat, a nonwoven mat, a mesh, a fabric, a sponge, a foam, a gel, a hydrogel, a tube, a particle (e.g., nano- or micro-particles, gel-like particles), a powder, a scaffold, a 3D construct, a coating layer on a substrate, or any combination thereof.

Methods for producing different forms of silk-based materials are known in the art, including but not limited to, for example, drying, solution casting, salt leaching, freeze-drying, gas formation, electrospinning, gelation (e.g., electrogelation), shear stress, sonication, pH reduction, water annealing, steam annealing, alcohol soaking, fiber drawing, coating, spraying, micronizing, or any combination thereof. In some specific embodiments, the silk fibroin composition (e.g., low molecular weight composition and/or stabilizing composition) is produced by a method comprising druing a solution of silk fibroin, optionally together with an activator/sample/ingredient; in some embodiments, the drying comprises lyophilization or air-drying.

In some embodiments, for example, depending on the water content remaining in the silk-based material, in some embodiments, the silk-based material (e.g., solid-state) can comprise at least about 10 wt % silk fibroin. For example, the silk-based material (e.g., solid-state) can comprise at least about 20 wt %, at least about 30 wt %, at least about 40 wt %, at least about 50 wt %, at least about 60 wt %, at least about 70 wt %, at least about 80 wt %, at least about 90 wt %, at least about 95 wt % silk fibroin.

In some embodiments, the silk fibroin compositions described herein (e.g., low molecular weight silk fibroin compositions and/or stabilized silk compositions) are in the form of particles. For example, the silk fibroin articles can be in the form of silk nanospheres or silk microspheres. As used herein, the term "particle" includes spheres; rods; shells; prisms; and powders, and in some embodiments, these particles can be part of a network or an aggregate. Without limitation, the particles can have any size from nanometers (nm) to millimeters (mm). In some embodiments, the particles can have a size in the range of from about 0.01 μm to about 1000 μm, from about 0.05 μm to about 500 μm, from about 0.1 μm to about 250 μm, from about 0.25 μm to about 200 μm, or from about 0.5 μm to about 100 μm. As used herein, the term "nanoparticle" refers to particles having a particle size of from about 0.1 nm to about 1000 nm. Certain embodiments of micro- to nano-scale silk fibroin particles and related techniques are also provided in U.S. provisional application filed concurrently herewith entitled "SYNTHESIS OF SILK FIBROIN MICRO- AND SUBMICRON SPHERES USING A CO-FLOW METHOD."

It will be understood by those skilled in the art that particles exhibit a distribution of particle sizes primarily around the above "size". Unless otherwise stated, the term "particle size" as used herein refers to the mode of the size distribution of a particle, i.e., the value that occurs predominantly in the size distribution. Methods for measuring particle size are known to those skilled in the art, for example, by dynamic light scattering (e.g., photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), and intermediate-angle laser light scattering (MALLS), light scattering methods (e.g., Coulter analytical methods), or other techniques (e.g., rheology, and light or electron microscopy).

In some embodiments, the particles can be substantially spherical. By "substantially spherical" is meant that the ratio of the length of the longest to shortest vertical axis of the particle cross-section is less than or equal to about 1.5. Substantially spherical does not require lines of symmetry. Additionally, the particles can have surface texturing, such as lines or indentations or protuberances that are small in size compared to the overall size of the particle and are still substantially spherical. In some embodiments, the ratio of the length between the longest and shortest axes of the particle is less than or equal to about 1.5, less than or equal to about 1.45, less than or equal to about 1.4, less than or equal to about 1.35, less than or equal to about 1.30, less than or equal to about 1.25, less than or equal to about 1.20, less than or equal to about 1.15, or less than or equal to about 1.1. Without wishing to be bound by theory, it is believed that surface contact is minimized with substantially spherical particles, which minimizes undesirable agglomeration of the particles during storage. Many crystals or flakes have flat surfaces that allow for large surface contact areas where aggregation can occur by ionic or non-ionic interactions. Spheres allow contact over a much smaller area.

In some embodiments, the particles have substantially the same particle size. Particles having a broad size distribution with both relatively new large and small particles allow the smaller particles to fill the spaces between the larger particles, thereby creating new contact surfaces. A broad size distribution provides many contact opportunities for agglomeration, resulting in larger spheres. The particles described herein have a narrow size distribution, thereby minimizing the opportunity for agglomeration. A "narrow size distribution" is a particle size distribution having a ratio of a volume diameter that is less than or equal to the 90th percentile 5 of the volume diameter of the small spherical particles relative to the 10th percentile of the volume diameter. In some embodiments, the 90th percentile volume diameter of the small spherical particles relative to the 10th percentile volume diameter is less than or equal to 4.5, less than or equal to 4, less than or equal to 3.5, less than or equal to 3, less than or equal to 2.5, less than or equal to 2, less than or equal to 1.5, less than or equal to 1.45, less than or equal to 1.40, less than or equal to 1.35, less than or equal to 1.3, less than or equal to 1.25, less than or equal to 1.20, less than or equal to 1.15, or less than or equal to 1.1.

Geometric standard deviation (GSD) can also be used to indicate a narrow size distribution. Calculating the GSD involves determining the effective intercept diameter (ECD) at accumulations less than 15.9% and 84.1%. The GSD is equal to the square root of the ratio of the ECD less than 84.17% to the ECD less than 15.9%. When the GSD is less than 2.5, the GSD has a narrow size distribution. In some embodiments, the GSD is less than 2, less than 1.75, or less than 1.5. In one embodiment, the GSD is less than 1.8.

Without wishing to be bound by theory, particle size can significantly determine the microscopic and macroscopic properties of the final product. Particle size depends on a number of process parameters, including but not limited to the size of the ceramic balls used, the amount of silk placed in each ball mill cup, the rotational speed (RPM) of the machine, and the duration of the ball milling. Particle size in the powder can be predicted based on mathematical modeling and/or experiments to determine correlations between some of these process parameters, for example. For example, this can be done by milling a given volume of silk fibroin for a variety of ball mill speeds and durations. Scanning electron microscopy (SEM) can be performed on representative samples from each experiment to measure particle size. Additional experiments can be run on each sample to determine the effect of the process parameters on the color, molecular weight, viscosity in solution, and water solubility of the resulting construct.

A variety of methods for producing silk particles (e.g., nanoparticles and microparticles) are known in the art. In some embodiments, the silk particles can be produced by a polyvinyl alcohol (PVA) phase separation method, for example, as described in International Application No. WO 2011/041395, the contents of which are herein incorporated by reference in their entirety. Other methods for producing silk fibroin particles are described, for example, in U.S. Application Publication No. U.S. 2010/0028451 and PCT Application Publication No. WO 2008/118133 (using lipids as templates to make silk microspheres or nanospheres), and Wenk et al., J Control Release, 2008; 132: 26-34 (using a spray method to produce silk microspheres or nanospheres), the entire contents of which are herein incorporated by reference in their entirety.

Without limitation, there are at least six types of particles that can be formulated with silk fibroin and an additive or active agent/sample: (1) particles comprising a core formed by silk fibroin, wherein the additive/agent/sample absorbs/adsorbs or forms a coating on the particle core; (2) particles comprising a core formed by an additive/agent/sample coated with one or more layers of silk fibroin; (3) particles comprising a generally homogeneous mixture of silk fibroin and an additive/agent/sample; (4) particles comprising a core comprising a mixture of silk fibroin and an additive/agent/sample having a coating of the core of silk fibroin; (5) particles comprising a core of silk fibroin or a material other than a biological sample, coated with one or more layers comprising a biological sample or silk fibroin or any combination of a biological sample and silk fibroin; (6) A particle comprising any of the particles of (1)-(5), and further comprising one or more layers of silk fibroin or a biological sample, e.g., a material other than a polymer. Silk fibroin particles (e.g., microspheres, nanospheres, or gel-like particles) and methods of making them are described, for example, in U.S. Pat. No. 8,187,616; and U.S. Patent Application Publications US 2008/0085272, US 2010/0028451, US 2012/0052124, US 2012/0070427, US 2012/0187591, which are incorporated herein by reference. Without limitation, the biological sample may be distributed in the silk fibroin matrix of the film, present on a surface of the film, coated on the film, or any combination thereof.

In some embodiments, the silk particles can be formed using the freeze-drying process described in U.S. Provisional Application No. 61/719,146, filed October 26, 2012, which is incorporated herein by reference in its entirety. Specifically, the silk foam can be formed by freeze-drying a silk solution. The foam can then be reduced to particles. For example, the silk solution can be cooled to a temperature at which the liquid carrier is transformed into a plurality of solid crystals or particles, and at least some of the plurality of solid crystals or particles can be removed, leaving a porous silk material (e.g., a silk foam). After cooling, the liquid carrier can be removed, at least partially, by sublimation, evaporation, and/or lyophilization. In some embodiments, the liquid carrier can be removed under reduced pressure. After formation, the silk fibroin foam can be subjected to pulverization, cutting, shredding, or any combination thereof to form silk particles. For example, the silk fibroin foam can be blended in a conventional blender or milled in a ball mill to form silk particles of a desired size. Accordingly, in some embodiments, the low molecular weight silk fibroin composition is in the form of a lyophilized powder.

In some embodiments, the silk fibroin composition (e.g., article) provided can be in the form of a gel or hydrogel. The term "hydrogel" is used herein to mean a silk-based material that exhibits the ability to retain a significant portion of water or other liquid within its structure without dissolution. Exemplary methods for preparing silk fibroin gels and hydrogels include, but are not limited to, sonication, vortexing, pH titration, exposure to an electric field, solvent soaking, water annealing, steam annealing, and the like. Exemplary methods for preparing silk fibroin gels and hydrogels are described, for example, in PCT Publication Nos. WO 2005/012606, WO 2008/150861, WO 2010/036992, and WO 2011/005381, all of which are incorporated herein by reference in their entireties. Gels formed by exposure to an electric field are also referred to herein as e-gels. Methods for forming e-gels are described, for example, in U.S. Patent Application Publication No. US2011/0171239, the entire contents of which are incorporated herein by reference. Without limitation, the additives/agents/samples may be distributed in the silk fibroin matrix of the gel or hydrogel, absorbed onto the surface of the gel or hydrogel or sponge, present within the pores of the gel or hydrogel, or any combination thereof.

In some embodiments, the silk fibroin compositions (e.g., articles) described herein can be in the form of a sponge or foam. In some embodiments, the foam or sponge can be a patterned foam or sponge, for example, a nanopatterned foam or sponge. Exemplary methods for making silk foams and sponges are described, for example, in PCT Application Publications WO 2004/000915, WO 2004/000255, and WO 2005/012606, the contents of which are incorporated herein by reference in their entirety. Without limitation, the additive/active/sample can be distributed in the silk fibroin matrix of the foam or sponge, absorbed onto the surface of the foam or sponge, present within the pores of the foam or sponge, or any combination thereof.

In some embodiments, the silk fibroin compositions (e.g., articles) described herein are in the form of fibers. As used herein, the term "fiber" means a unit of material that is relatively flexible and has a high ratio of length to width across its cross-section perpendicular to its length. Methods for making silk fibroin fibers are well known in the art. Fibers can be made by electrospinning a silk solution, drawing a silk solution, and the like. Electrospun silk materials, such as fibers, and methods for making them are described in, for example, WO2011/008842, the entire contents of which are incorporated herein by reference. Micron-sized silk fibers (e.g., 10-600 μm in size) and methods for making them are described in, for example, Mandal et al., PNAS, 2012, doi: 10.1073/pnas.1119474109; U.S. Provisional Application No. 61/621,209, filed April 6, 2012; and PCT Application No. PCT/US13/35389, filed April 5, 2013.

In some embodiments, the silk fibroin composition (e.g., article) described herein can be in the form of a film, e.g., a silk film. As used herein, the term "film" refers to a substantially flat structure, and can also include structures formed from a substantially flat structure. For example, the term "film" can include a tubular structure or any structure that can be formed by manipulating (e.g., rolling up and/or folding) a substantially flat film into a desired shape. The term "film" is used in a comprehensive sense, including webs, sheets, laminates, and the like. In some embodiments, the film is a patterned film, e.g., a nanopatterned film. Exemplary methods for making silk fibroin films are described, for example, in PCT Application Publication Nos. WO 2004/000915 and WO 2005/012606, both of which are incorporated herein by reference in their entireties. In some embodiments, when additives and/or agents/samples are included, they may be distributed in the film, present on the surface of the film, coated on the film, or any combination thereof.

The film can have any desired thickness. For example, the film thickness can range from about 1 nm to about 10 mm. In some embodiments, the film has a thickness in the range from about 1 nm to about 1000 nm or from about 1 μm to about 1000 μm.

In some embodiments, the silk fibroin compositions described herein can be in the form of a coating layer. In some embodiments, such a coating layer can be on a substrate surface. Without limitation, the silk fibroin coating layer can comprise one or more layers. Additionally, if there are two or more layers, each layer can be a single layer comprising the silk fibroin composition (e.g., low molecular weight silk fibroin) or multiple layers, wherein at least one or more layers comprises low molecular weight silk fibroin fragments. In some embodiments, the coating layer is a very thin coating layer. In some embodiments, the silk fibroin compositions disclosed herein can form a portion of the substrate.

Examples of substrates may include, but are not limited to, dipsticks, cellulose-based products, micro-titer plates, specimen containers (such as, but not limited to, blood collection containers), medical devices, grafts, and any combination thereof. A method of forming a coating layer comprising silk fibroin is described in U.S. patent application Ser. No. 11/997,193, the entire contents of which are incorporated herein by reference.

In some embodiments, the silk fibroin compositions (e.g., articles) described herein can be in the form of a cylindrical matrix, e.g., a silk tube. The silk tube can be made using any method known in the art. For example, the tube can be made using molding, dipping, electrospinning, gel spinning, and the like. Gel spinning is described in Lovett et al., Biomaterials 2008, 29(35):4650-4657, both of which are incorporated herein by reference in their entirety, and structures of gel-spun silk tubes are described in PCT Application No. PCT/US2009/039870, filed April 8, 2009, the contents of which are both incorporated herein by reference in their entirety. Structures of silk tubes using a dip-coating method are described in PCT Application No. PCT/US2008/072742, filed August 11, 2008, the contents of which are incorporated herein by reference in their entirety. Structures of silk fibroin tubes using film-spinning methods are described in PCT Application No. PCT/US2013/030206, filed March 11, 2013; U.S. Provisional Application No. 61/613,185, filed March 20, 2012; and PCT Application Publication No. WO 2013126799, both of which are hereby incorporated by reference in their entireties. Without wishing to be bound by theory, it is believed that the inner and outer diameters of the silk tubes can be more easily controlled using film-spinning or gel-spinning than by dip-coating techniques.

In some embodiments, the silk-based material provided is in the form of a matrix comprising a lumen or cavity therein. In some such embodiments, at least a portion of the additive and/or active agent/sample is distributed within the silk fibroin network. In some embodiments, this may be present in the lumen or cavity. In some embodiments, the silk fibroin is in the form of a matrix comprising a lumen or cavity therein, and at least a portion of the additive/agent/sample is distributed within the silk fibroin network itself. In some embodiments, when the matrix comprises a lumen or cavity, at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) of the additive or agent may be present in the lumen or cavity. In some embodiments, the entire amount is present in the lumen/cavity. In some embodiments, at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) of the silk fibroin network of the matrix (e.g., the non-lumenal portion of the silk-based material) can be present.

In some embodiments, the silk fibroin compositions (e.g., articles) disclosed herein can be porous (e.g., porous matrices or scaffolds). For example, the porous scaffold can have a porosity of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more. As used herein, the term "porosity" is a measure of the void space in a material, as a percentage from 0 to 100% (or from 0 to 1), and is the fraction of the volume of voids over the total volume. Measurements of porosity are well known to those of ordinary skill in the art, for example, using standardized techniques such as mercury porosimetry and gas adsorption, such as nitrogen adsorption. As used herein, the term "porosity" is a measure of the void space in a material, as a percentage from 0 to 100% (or from 0 to 1), and is the fraction of void volume over the total volume. Measurements of porosity are well known to those of ordinary skill in the art, for example, using standardized techniques such as mercury porosimetry and gas adsorption, such as nitrogen adsorption.

The porous scaffold can have any pore size. As used herein, the term "pore size" refers to the diameter of a cross-section of a pore or an effective diameter. The term "pore size" can also refer to the average diameter of a cross-section of a pore or an average effective diameter based on measurements of a plurality of pores. The effective diameter of a cross-section is not the same as the diameter of a circular cross-section having the same cross-sectional area as that of a non-circular cross-section.

In some embodiments, the pores of the silk-based material can have a size distribution in the range of from about 50 nm to about 1000 nm, from about 250 nm to about 500 nm, from about 500 nm to about 250 nm, from about 1 nm to about 200 nm, from about 10 nm to about 150 nm, or from about 50 nm to about 100 nm. In some embodiments, the silk-based material can be swellable when hydrated. The size of the pores can then vary depending on the water content within the silk-based material. In some embodiments, the pores can be filled with a fluid such as water or air.

Methods for forming pores within silk fibroin-based scaffolds are known in the art and include, but are not limited to, porogen-leaching methods, freeze-drying methods, and/or gas-forming methods. Exemplary methods for forming pores within silk-based materials are described, for example, in U.S. Patent Application Publication Nos. US 2010/0279112 and US 2010/0279112; U.S. Patent No. 7,842,780; and WO2004062697, the entire contents of which are incorporated herein by reference.

Without wishing to be bound by theory, it is believed that the porosity, structure, and mechanical properties of the porous scaffold can be controlled via different post-spinning methods, such as steam annealing, heat treatment, alcohol treatment, air-drying, lyophilization, etc. Additionally, any desired release rate, profile, or kinetics of the encapsulated molecules within the matrix can be varied by varying processing parameters, such as matrix thickness, silk molecular weight, silk concentration within the matrix, beta-sheet conformational structure, silk II beta-sheet crystallinity, or porosity and pore size. In some embodiments, low molecular weight silk fibroin scaffolds can provide sufficient mechanical strength/stability at the implant site while still allowing the scaffold to degrade over a desired period of time (e.g., a month or more). Without wishing to be bound by theory, in some embodiments, the porosity of the silk-based material can be controlled for a desired dissolution rate. For example, a higher porosity of a silk-based material can generally allow an aqueous solution to penetrate more quickly into the silk-based material, thus accelerating the dissolution process. One skilled in the art can appropriately adjust the porosity based on a number of factors, including but not limited to the desired dissolution rate; the molecular size and/or diffusion coefficient of the components present in the silk-based material; and/or the concentration, amount, and/or desired physical or mechanical properties of the silk-based material.

To incorporate additives/agents/samples into the silk fibroin matrix, these may be included in the silk fibroin solution used to produce the silk-based material. Alternatively, or additionally, a pre-formed silk-based material may be added to the solution containing the additives/agents/samples, such that the additives/agents/samples are absorbed into/upon the silk-based material.

In some embodiments, the additive/agent/sample can be dispersed uniformly or non-uniformly (e.g., having a gradient) within the silk-based material. In some embodiments, the additive/agent/sample can be encapsulated or sealed within the silk-based material by the silk fibroin. In some embodiments, the additive/agent/sample can be mixed or blended with the silk fibroin within the silk-based material.

In some embodiments, the silk fibroin compositions disclosed herein are osteoconductive. Osteoconductivity is generally defined as the ability of a material to facilitate the migration of osteogenic cells to the surface of a scaffold via fibrin clots that form immediately after the material is implanted. The porosity of a material affects the osteoconductivity of that material.

In some embodiments, the silk fibroin compositions disclosed herein are osteoinductive. Osteoinductiveness is generally defined as the ability to induce undifferentiated stem cells or osteoprogenitor cells (osteoblasts), which are components of bone (bone) tissue, to differentiate into osteoblasts. The simplest test of osteoinductiveness is the ability to induce bone formation in tissue sites that do not normally form bone, such as muscle (normal bone growth). It is generally understood that articles of manufacture disclosed herein can be made osteoinductive by adding growth factors thereto, such as rhBMP-2 (recombinant human bone morphogenetic protein-2). Mineralization and the addition of growth factors can affect the osteoinductiveness of the material.

In some embodiments, the silk fibroin compositions disclosed herein are osteogenic and exhibit new bone formation following in vivo implantation. Osteogenesis is the process of laying down new bone material using osteoblasts. Osteoblasts create bone by forming an osteoid matrix, which is composed primarily of type I collagen. Osteogenesis comprises minerals (mostly calcium phosphate) that form the osteoid matrix and a chemical arrangement called calcium hydroxyapatite. Osteoblasts are typically responsible for mineralizing the osteoid matrix to form bone tissue. Without wishing to be bound by theory, the osteoconductivity and osteoinductive properties of the material influence osteogenesis. The material can exhibit new bone formation within 6 months following in vivo implantation, and in some embodiments, exhibit new bone formation within 8 weeks following in vivo implantation.

In some embodiments, the silk fibroin articles disclosed herein can be sterilized using conventional sterilization processes, such as radiation sterilization (i.e., gamma rays), chemical sterilization (ethylene oxide), autoclaving, or other suitable procedures. In some embodiments, the sterilization process can utilize ethylene oxide at a temperature of between about 52° C. and about 55° C. for a time of up to 8 hours. In some embodiments, the silk fibroin articles disclosed herein can also be sterilized. The sterilized silk fibroin articles disclosed herein can be packaged in a suitable sterilization moisture-resistant package for shipping. In some embodiments, the silk fibroin composition containing the active agent is not subjected to sterilization that significantly damages or degrades the active agent. In some embodiments, the silk fibroin composition protects the active agent from sterilization hazards.

In some embodiments, the silk fibroin article disclosed herein is in the form of an implant or implantable drug delivery device.

In some embodiments, the silk fibroin composition provided is in the form of an injectable composition. The term "injectable composition" as used herein generally refers to a composition that can be delivered or administered into a tissue by a minimally invasive procedure. The term "minimally invasive procedure" refers to a procedure that enters the body or skin of a subject or a body cavity or an anatomical opening but is performed with the least possible damage (e.g., a small incision, an injection). In some embodiments, the injectable composition can be administered or delivered into a tissue by injection. In some embodiments, the injectable composition can be administered or delivered into a tissue through a small incision in the skin following the insertion of a needle, cannula, and/or tubing, e.g., a catheter. Without wishing to be limited thereto, the injectable composition can be administered or placed into a tissue by surgery, e.g., by implantation. Some exemplary injectable compositions include, but are not limited to, solutions, hydrogels, gel-like particles, and/or microspheres.

To be clear, the term "injectable" in "injectable formulation" and "injection" means the physical characteristics of a solution (e.g., formulation) that are suitable for administration by injection, such that a flow rate of solution is sufficient to pass through a needle or any other suitable means, and such flow rate can be reasonably conveniently generated by a user. Syringes are commonly used to deliver injectable drugs to a subject. In some embodiments, the injectable formulation may be provided in a pre-filled syringe. In some embodiments, the injectable formulation may be provided as a ready-to-use formulation. In some embodiments, the injectable formulation may be provided in a kit.

In some embodiments, the composition provided, e.g., an injectable composition, can further comprise a pharmaceutically acceptable carrier. For example, in some embodiments, the composition suitable for injection comprises a sterile aqueous solution or dispersion. The carrier can be a solvent or dispersion medium, including, for example, water, cell culture medium, a buffer (e.g., phosphate buffered saline), a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), a suitable mixture thereof. In some embodiments, the pharmaceutical carrier can be a buffered solution (e.g., PBS).

Alternatively or additionally, various additives such as antimicrobial preservatives, antioxidants, chelating agents, and buffers may be added to increase the stability, sterility, and isotonicity of the injectable composition. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it may be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. The injectable composition may also contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, dyes, and the like, depending on the purpose of manufacture.

The viscosity of the provided liquid composition (e.g., an injectable composition) can be controlled by controlling the weight percentage of silk fibroin fragments having molecular weight sub-ranges (i) to (xviii). In some embodiments, the viscosity of the composition can be further maintained at a selected level using a pharmaceutically acceptable thickener. In one embodiment, methylcellulose can be used because it is readily and economically available and easy to work with. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The desired concentration of the thickener can vary depending on the selected agent and the desired viscosity for the injection. The important thing is to use an amount that will achieve the selected viscosity, for example, to add such a thickener to the injectable composition of some embodiments.

In some embodiments, the provided silk fibroin composition is in the form of a sprayable composition.

In some embodiments, the silk fibroin composition provided herein (e.g., a low molecular weight silk composition) can be in the form of an aerogel or aerogel-like material. For example, methods of forming an aerogel or aerogel-like material comprising silk fibroin are described in U.S. Provisional Patent Application No. US 61/902,145, filed November 8, 2013, entitled "Peptide-Based Nanofibril Materials," which is herein incorporated by reference in its entirety.

Without wishing to be bound by theory, it is believed that the properties of the silk-based material (including but not limited to, solubility) (including, but not limited to, the active agents or biological samples described herein) can be altered by varying the molecular weight of the silk fibroin fragments within the silk-based material. In some embodiments, different molecular weights of silk fibroin can be produced by using different periods of time to refine the cocoons to provide the refined fibroin. Thus, in some embodiments for producing low molecular weight silk fibroin compositions, the cocoons are boiled (e.g., in a salt solution, such as Na 2 CO 3 ) for a period of time between about 1 minute and 2 hours, between about 5 minutes and 2 hours, between about 10 minutes and 60 minutes, between about 60 minutes and 4 hours, between about 60 minutes and 3 hours, between about 60 minutes and 2 hours, between about 60 minutes and ₉₀ minutes, or about ₄ hours or more. In some embodiments, the boiling can be carried out for about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 100 minutes or more (e.g., in a salt solution, such as Na ₂ CO ₃ ). By controlling the boiling time, the solubility of the silk-based material can be optimized for extraction or recovery of agents present in the low molecular weight silk fibroin composition (e.g., in aqueous solution). Without wishing to be bound by theory, it is believed that longer boiling times generally yield silk fibroin of lower molecular weight (MW)/chain length, and thus, silk-based materials produced from lower MW silk fibroin may generally be more soluble (e.g., in aqueous solution) than those produced from higher MW silk fibroin.

In some embodiments, the soluble silk-based material can be produced from a silk solution that has been boiled or scoured for a period of time sufficient to yield low MW silk fibroin therein, for example, at least about 30 minutes, at least about 60 minutes, or at least about 90 minutes or more. In some embodiments, the soluble silk-based material is a lyophilized silk-based material produced from a silk solution that has been boiled or scoured for at least about 30 minutes, at least about 60 minutes, or at least about 90 minutes or more. In some embodiments, the soluble silk-based material is a silk-based film produced from a silk solution that has been boiled or scoured for at least about 30 minutes, at least about 60 minutes, or at least about 90 minutes or more. The silk-based material can be produced in any of the other forms described herein, for example, by electrospinning, gelling or other rapid solidification techniques known in the art.

Other methods for modifying the solubility of the silk-based material described herein may also be used alone or in combination with controlling the refining time. For example, the solubility of the silk-based material may be improved by methods that accelerate the drying rate of the silk-based material, such as pressurized air, reduced humidity (less than ambient), and/or increased temperature, etc. Additionally or alternatively, the solubility of the silk-based material may be improved by decreasing the time that the silk-based material is subjected to lyophilization conditions and/or exposed to conditions that induce crystallization within the silk fibroin. In some embodiments, the solubility of the silk-based material may be improved by selectively removing the high molecular weight fraction of the silk (e.g., heavy chains and/or long hydrophobic sequences) during purification, such as by enzymatic digestion, filtration, or chromatography. In some embodiments, the solubility of the silk-based material can be improved by sterilization means, such as autoclaving and sterile filtration, which function to further reduce molecular weight and/or remove insoluble particulates.

In various embodiments, at least a portion of the silk fibroin in the silk fibroin compositions described herein (e.g., in the low molecular weight silk fibroin compositions and/or in the stabilized compositions) can be modified for different uses, e.g., biomedical applications, and/or for different desired mechanical or chemical properties. The skilled person can select an appropriate method to modify the silk fibroin depending on, for example, the side groups of the silk fibroin, the desired reactivity in the silk fibroin, and/or the desired charge density on the silk fibroin.

For example, to maintain the stability of an active agent dispersed in the silk fibroin composition when implanted in vivo for tissue engineering or drug delivery purposes, at least a portion of the silk fibroin can be genetically modified to provide for additional modifications of the silk, such as inclusion of a fusion polypeptide comprising a fibrous protein domain and a mineralizing domain that can be used to form organic-inorganic composites. See, e.g., WO 2006/076711, which is herein incorporated by reference in its entirety. In some embodiments, the silk fibroin can be chemically modified, such as by diazonium or carbodiimide coupling reactions, avidin-biotin interactions, or genetically modified, to change the physical properties and functionality of the silk protein. Chemically modified silk fibroin and methods for making them are described in, e.g., PCT Application Publication Nos. WO2011/011347 and WO2010/057142; and described in U.S. Patent Application No. 12/192,588.

In one embodiment, the modification of silk fibroin can utilize amino acid side chain chemistry, such as chemical modification or covalent bonding, or modification or charge-charge interactions. Exemplary chemical modification methods include, but are not limited to, carbodiimide coupling reactions (see, e.g., U.S. Patent Application No. US 2007/0212730 ), diazonium coupling reactions (see, e.g., U.S. Patent Application No. US 2009/0232963 ), avidin-biotin interactions (see, e.g., International Application No. WO 2011/011347 ), and PEGylation using chemically active or activated derivatives of PEG polymers (see, e.g., International Application No. WO 2010/057142 ). Silk fibroin can also be modified via genetic modification to alter the functionality of the silk protein. (see, e.g., International Application No. WO 2011/006133 ). For example, silk fibroin can be genetically modified to provide for additional modifications of the silk, such as inclusion of a fusion polypeptide comprising a fibrous protein domain and a mineralizing domain, which can be used to form organic-inorganic composites. See WO 2006/076711. In some embodiments, silk fibroin can be genetically modified to fuse with a protein, such as a therapeutic protein.

In some embodiments, at least a portion of the silk fibroin in the compositions described herein can be derivatized or modified with a positively/negatively charged molecule. In some embodiments, the silk fibroin can be modified with a positively/negatively charged peptide or polypeptide, such as poly-lysine and poly-glutamic acid. Although possible, it is not required that every single silk fibroin molecule in the composition be modified with a positively/negatively charged molecule. For example, methods of derivatizing or modifying silk fibroin with a charged molecule are described in PCT Application Publication No. WO2011109691A2, the entire contents of which are incorporated herein by reference.

The ratio of modified silk fibroin to unmodified silk fibroin can be adjusted to optimize one or more desired properties of the low molecular weight silk fibroin composition or article formed therefrom. Thus, in some embodiments, the ratio of modified silk fibroin to unmodified silk fibroin in the composition can range from about 1000:1 (w/w) to about 1:1000 (w/w), from about 500:1 (w/w) to about 1:500 (w/w), from about 250:1 (w/w) to about 1:250 (w/w), from about 200:1 (w/w) to about 1:200 (w/w), from about 25:1 (w/w) to about 1:25 (w/w), from about 20:1 (w/w) to about 1:20 (w/w), from about 10:1 (w/w) to about 1:10 (w/w), or from about 5:1 (w/w) to about 1:5 (w/w).

In some embodiments, the composition comprises, for example, at least 1000:1, at least 900:1, at least 800:1, at least 700:1, at least 600:1, at least 500:1, at least 400:1, at least 300:1, at least 200:1, at least 100:1, at least 90:1, at least 80:1, at least 70:1, at least 60:1, at least 50:1, at least 40:1, at least 30:1, at least 20:1, at least 10:1, at least 7:1, at least 5:1, at least 3:1, at least 1:1, at least 1:3, at least 1:5, at least 1:7, at least 1:10, at least 1:20, at least 1:30, at least 1:40, at least 1:50, at least 1:60, at least 1:70, A molar ratio of modified silk fibroin to unmodified silk fibroin of at least 1:80, at least 1:90, at least 1:100, at least 1:200, at least 1:300, at least 1:400, at least 1:500, at least 600, at least 1:700, at least 1:800, at least 1:900, or at least 1:100.

In some embodiments, the composition comprises, for example, at most 1000:1, at most 900:1, at most 800:1, at most 700:1, at most 600:1, at most 500:1, at most 400:1, at most 300:1, at most 200:1, 100:1, at most 90:1, at most 80:1, at most 70:1, at most 60:1, at most 50:1, at most 40:1, at most 30:1, at most 20:1, at most 10:1, at most 7:1, at most 5:1, at most 3:1, at most 1:1, at most 1:3, at most 1:5, at most 1:7, at most 1:10, at most 1:20, at most 1:30, at most 1:40, at most 1:50, at most 1:60, at most 1:70, A molar ratio of modified silk fibroin to unmodified silk fibroin of at most 1:80, at most 1:90, at most 1:100, at most 1:200, at most 1:300, at most 1:400, at most 1:500, at most 1:600, at most 1:700, at most 1:800, at most 1:900, or at most 1:1000.

In some embodiments, the composition comprises, for example, about 1000:1 to about 1:1000, about 900:1 to about 1:900, about 800:1 to about 1:800, about 700:1 to about 1:700, about 600:1 to about 1:600, about 500:1 to about 1:500, about 400:1 to about 1:400, about 300:1 to about 1:300, about 200:1 to about 1:200, about 100:1 to about 1:100, about 90:1 to about 1:90, about 80:1 to about 1:80, about 70:1 to about 1:70, about 60:1 to about 1:60, about 50:1 to about 1:50, about A molar ratio of modified silk fibroin to unmodified silk fibroin of about 40:1 to about 1:40, about 30:1 to about 1:30, about 20:1 to about 1:20, about 10:1 to about 1:10, about 7:1 to about 1:7, about 5:1 to about 1:5, about 3:1 to about 1:3, or about 1:1.

In some embodiments, the silk fibroin is substantially deficient in its original sericin content (e.g., less than or equal to 5% (w/w) residual sericin in the final extracted silk). Alternatively, more concentrated residual sericin may remain on the silk after extraction, or the extraction step may be omitted. In some embodiments, the sericin deficient silk fibroin has, for example, about 1% (w/w) residual sericin, about 2% (w/w) residual sericin, about 3% (w/w) residual sericin, about 4% (w/w), or about 5% (w/w) residual sericin. In some embodiments, the sericin-deficient silk fibroin has, for example, at most 1% (w/w) residual sericin, at most 2% (w/w) residual sericin, at most 3% (w/w) residual sericin, at most 4% (w/w), or at most 5% (w/w) residual sericin. In some other embodiments, the sericin deficient silk fibroin has, for example, about 1% (w/w) to about 2% (w/w) residual sericin, about 1% (w/w) to about 3% (w/w) residual sericin, about 1% (w/w) to about 4% (w/w), or about 1% (w/w) to about 5% (w/w) residual sericin. In some embodiments, the silk fibroin is completely free of its original sericin content. As used herein, the term "completely free" (i.e., "consisting of") means that the substance cannot be detected or its presence is not determined within the detection range of the device or the process used. In some embodiments, the silk fibroin is substantially free of its original sericin content. As used herein, the term "substantially free" (or "consisting of") means that only trace amounts of the substance can be detected, and that less than or equal to a detectable amount of the substance is present.

Without wishing to be bound by theory, the properties of the silk fibroin compositions provided may be modified by controlled partial removal of silk sericin or by intentional enrichment of sericin in the source silk. This may be achieved by modifying conditions during the silk refining process, such as time, temperature, concentration, etc.

The refined silk can be prepared by any conventionally known method known to those of ordinary skill in the art. For example, the silkworm cocoons are boiled in an aqueous solution for a predetermined period of time. Typically, longer refining times result in lower molecular weight silk fibroin. In some embodiments, the cocoons are boiled for at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, or more to produce lower molecular weight silk fibroin fragments. Additionally or alternatively, in some embodiments, the cocoons can be heated or boiled at an elevated temperature. For example, in some embodiments, at about 101.0°C, at about 101.5°C, at about 102.0°C, at about 102.5°C, at about 103.0°C, at about 103.5°C, at about 104.0°C, at about 104.5°C, at about 105.0°C, at about 105.5°C, at about 106.0°C, at about 106.5°C, at about 107.0°C, at about 107.5°C, at about 108.0°C, at about 108.5°C, at about 109.0°C, at about 109.5°C, at about 110.0°C, at about 110.5°C, at about 111.0°C, at about 111.5°C, at about 112.0°C, at about The cocoon can be heated or boiled at about 112.5°C, about 113.0°C, about 113.5°C, about 114.0°C, about 114.5°C, about 115.0°C, about 115.5°C, about 116.0°C, about 116.5°C, about 117.0°C, about 117.5°C, about 118.0°C, about 118.5°C, about 119.0°C, about 119.5°C, about 120.0°C, or higher. In some embodiments, such heating can be achieved by performing at least part of the heating process (e.g., boiling process) under pressure. For example, suitable pressures at which the silk fibroin fragments described herein can be produced are typically between about 10-40 psi, for example, about 11 psi, about 12 psi, about 13 psi, about 14 psi, about 15 psi, about 16 psi, about 17 psi, about 18 psi, about 19 psi, about 20 psi, about 21 psi, about 22 psi, about 23 psi, about 24 psi, about 25 psi, about 26 psi, about 27 psi, about 28 psi, about 29 psi, about 30 psi, about 31 psi, about 32 psi, about 33 psi, about 34 psi, about 35 psi, about 36 psi, about 37 psi, about 38 psi, about 39 psi, or about 40 psi.

In one embodiment, the aqueous solution used in the process of refining the silkworm cocoons is about 0.02 M Na ₂ CO ₃ . The cocoons are washed, for example, with water, to extract the sericin protein. The refined silk can be dried and used to prepare a silk powder. Alternatively, the extracted silk can be dissolved in an aqueous salt solution. Useful salts for this purpose include lithium bromide, lithium thiocyanate, calcium nitrate, or other chemicals capable of dissolving the silk. In some embodiments, the extracted silk can be dissolved in about 8 M -12 M LiBr solution. The salt is subsequently removed, for example, using dialysis.

If necessary, the solution can then be concentrated using dialysis against, for example, a hygroscopic polymer, such as PEG, a polyethylene oxide, amylose or sericin. In some embodiments, the PEG has a molecular weight of 8,000-10,000 g/mol and a concentration of from about 10% to about 50% (w/v). A Slide-a-Lyzer dialysis cassette (Pierce, MW CO 3500) can be used. However, any dialysis system can be used. Dialysis can be performed for a period of time sufficient to obtain a final concentration of the aqueous silk solution of from about 10% to about 30%. In most cases, dialysis for 2 to 12 hours can be sufficient. See, for example, International Patent Application Publication No. WO 2005/012606, which is herein incorporated by reference in its entirety.

Another method for producing a concentrated silk solution involves drying a diluted silk solution (e.g., via evaporation or lyophilization). The diluted solution may be partially dried to reduce its volume, thereby increasing the silk concentration. The diluted solution may be completely dried, allowing the dried silk fibroin to be dissolved in a smaller volume of solvent compared to the diluted silk solution. In some embodiments, the silk fibroin solution may optionally be filtered and/or centrifuged at a suitable point. For example, in some embodiments, the silk fibroin solution may optionally be filtered and/or centrifuged followed by a heating or boiling step. In some embodiments, the silk fibroin solution may optionally be filtered and/or centrifuged followed by a dialysis step. In some embodiments, the silk fibroin solution may optionally be filtered and/or centrifuged followed by a concentration adjustment step. In some embodiments, the silk fibroin solution may optionally be filtered and/or centrifuged followed by a reconstitution step. In any of these embodiments, the filtration and/or centrifugation step(s) may be performed to remove insoluble material. In any of these embodiments, the filtration and/or centrifugation step(s) may optionally be performed to enrich the concentration of silk fibroin fragment(s) of a particular molecular weight.

In some embodiments, the silk fibroin solution can be produced using an organic solvent. Such methods have been described, for example, in Li, M., et al., J. Appl. Poly Sci. 2001, 79, 2192-2199; Min, S., et al. Sen'I Gakkaishi 1997, 54, 85-92; Nazarov, R. et al., Biomacromolecules 2004 5,718-26, the entire contents of which are incorporated herein by reference. Exemplary organic solvents that can be used to produce the silk solution include, but are not limited to, hexafluoroisopropanol (HFIP). See, for example, International Application No. WO2004/000915, the entire contents of which are incorporated herein by reference. In some embodiments, the silk solution is completely or substantially free from organic solvent; in some embodiments, it is substantially free from other solvents other than water.

The silk fibroin compositions disclosed herein can include any amount/ratio of silk fibroin to total volume/weight of the composition. Without wishing to be bound by theory, it is believed that the amount of silk fibroin in the solution used to make the silk fibroin composition (e.g., the low molecular weight silk fibroin composition and/or the stabilizing composition) can be varied, thereby changing the properties of the silk fibroin composition. Generally, any amount of silk fibroin can be present in the solution used to make the silk fibroin composition. For example, the amount of silk fibroin in the solution can be from about 0.1% (w/v) to about 90% (w/v). In some embodiments, the amount of silk fibroin in the solution is from about 1% (w/v) to about 75% (w/v), from about 1% (w/v) to about 70% (w/v), from about 1% (w/v) to about 65% (w/v), from about 1% (w/v) to about 60% (w/v), from about 1% (w/v) to about 55% (w/v), from about 1% (w/v) to about 50% (w/v), from about 1% (w/v) to about 35% (w/v), from about 1% (w/v) to about 30% (w/v), from about 1% (w/v) to about 25% (w/v), from about 1% (w/v) to about 20% (w/v), from about 1% (w/v) to about 15% (w/v), from about 1% (w/v) to about The silk fibroin in the solution can be about 10% (w/v), about 5% (w/v) to about 25% (w/v), about 5% (w/v) to about 20% (w/v), about 5% (w/v) to about 15% (w/v). In some embodiments, the silk fibroin in the solution is about 25% (w/v). In some embodiments, the silk fibroin in the solution is about 0.5 (w/v) to about 30% (w/v), about 4% (w/v) to about 16% (w/v), about 4% (w/v) to about 14% (w/v), about 4% (w/v) to about 12% (w/v), about 4% (w/v) to about 0% (w/v), about 6% (w/v) to about 8% (w/v). The exact amount of silk in a silk solution can be determined by drying a known amount of silk solution and measuring the mass of what remains to calculate the solution concentration.

The amount of silk fibroin in a provided composition or article (e.g., in a low molecular weight silk composition and/or a stabilizing composition) can be from about 1% (w/v) to about 90% (w/v). In some embodiments, the amount of silk fibroin in the silk fibroin composition is from about 0.1% (w/v) to about 75% (w/v), from about 1% (w/v) to about 70% (w/v), from about 1% (w/v) to about 65% (w/v), from about 1% (w/v) to about 60% (w/v), from about 1% (w/v) to about 55% (w/v), from about 1% (w/v) to about 50% (w/v), from about 1% (w/v) to about 45% (w/v), from about 1% (w/v) to about 40% (w/v), from about 1% (w/v) to about 35% (w/v), from about 1% (w/v) to about 30% (w/v), from about 1% (w/v) to about 25% (w/v), from about 1% (w/v) to about 20% (w/v), about 1% (w/v) to about 15% (w/v), about 1% (w/v) to about 10% (w/v), about 5% (w/v) to about 25% (w/v), about 5% (w/v) to about 20% (w/v), about 5% (w/v) to about 15% (w/v). In some embodiments, the silk fibroin in the low molecular weight silk fibroin composition is about 25% (w/v). In some embodiments, the silk in the silk fibroin composition is from about 0.5 (w/v) to about 30% (w/v), from about 2% (w/v) to about 8% (w/v), from about 2% (w/v) to about 7% (w/v), from about 2% (w/v) to about 6% (w/v), from about 2% (w/v) to about 5% (w/v), or from about 3% (w/v) to about 4% (w/v).

In some embodiments, the solution has a silk fibroin concentration of about 0.25% to about 50% (w/v), or about 0.5% to about 15% (w/v), or about 0.5% to about 10% (w/v). In some embodiments, the silk fibroin solution has a silk fibroin concentration of about 10% to about 40%, or 15% to about 35% (w/v). In one embodiment, the silk fibroin solution has a silk fibroin concentration of about 20% to about 30% (w/v). In one embodiment, the silk fibroin solution has a silk fibroin concentration of about 30% (w/v). In some embodiments, the silk fibroin solution has a silk fibroin concentration of about 0.1% to about 30% (w/v), about 0.5% to about 15% (w/v), about 1% to about 8% (w/v), or about 1.5% to about 5% (w/v). In some embodiments, the silk fibroin solution has a silk fibroin concentration of about 5% to about 30% (w/v), about 10% to about 25% (w/v), or about 15 to about 20% (w/v). In some embodiments, the silk solution has a silk fibroin concentration of about 0.5% to 10% (w/v).

In some embodiments, depending on the application, a conformational change within the silk fibroin in the provided composition can be induced to control the solubility of the silk fibroin composition/article. In some embodiments, the conformational change can cause the silk fibroin to become at least partially insoluble. Without wishing to be bound by theory, the induced conformational change changes the crystallinity of the silk fibroin, e.g., silk II beta-sheet crystallinity. The conformational change can be induced by any method known in the art, including alcohol immersion (e.g., ethanol, methanol), water annealing, shear stress, ultrasound (e.g., sonication), pH reduction (e.g., pH titration and/or exposure to an electric field), and any combination thereof. For example, the conformational change can be induced by slow drying (Lu et al., Biomacromolecules 2009, 10, 1032); Water annealing (reference [Jin et al., 15 Adv. Funct. Mats. 2005, 15, 1241], [Hu et al., Biomacromolecules 2011, 12, 1686)]; stretching (reference [Demura & Asakura, Biotech & Bioengin. 1989, 33, 598]); pressurization; Solvent immersion including methanol (Hofmann et al., J Control Release. 2006, 111, 219), ethanol (Miyairi et al., J. Fermen. Tech. 1978, 56, 303), glutaraldehyde (Acharya et al., Biotechnol J. 2008, 3, 226), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Bayraktar et al., Eur J Pharm Biopharm. 2005, 60, 373]); pH adjustment, e.g., pH titration and/or exposure to an electric field (see, e.g., US patent application Ser. No. US2011/0171239); heat treatment; can be induced by one or more methods including, but not limited to, shear stress (see, e.g., International Application No. WO2011/005381), ultrasound, e.g., sonication (see, e.g., U.S. Patent Application Publication No. U.S. 2010/0178304 and International Application No. WO2008/150861); and any combination thereof. The contents of all of the above references are incorporated herein by reference in their entirety.

In some embodiments, the material may be treated by annealing. The annealing process used herein involves inducing the formation of beta-sheet secondary structure within the silk fibroin. This may be due to enhanced non-covalent interactions within the silk fibroin. These non-covalent interactions may include intramolecular interactions, intermolecular interactions, or both. Typically, the non-covalent interactions may be mediated by hydrogen bonding, which leads to enhanced beta sheet formation. Upon reaching a certain critical level of beta sheet secondary structure, the silk fibroin becomes insoluble, for example, in an aqueous environment. This phenomenon is generally referred to as crystallinity, and this state of the silk fibroin is referred to as silk II. Thus, "annealing" involves a conformational change of the silk fibroin into a beta-sheet dominant (silk II) conformation, whereby the silk fibroin crystallizes and is therefore insoluble. Although we do not wish to be bound by theory, it is thought that these conformational changes are due to structural changes mediated by hydrogen-bonding and/or hydrophobic interactions toward a higher beta-sheet content of silk fibroin.

In some embodiments, the conformation of silk fibroin can be changed by water annealing. There are many different methods of water annealing. One method of water annealing involves treating silk fibroin in a solidified but soluble form with water vapor. Without wishing to be bound by theory, it is thought that the water molecules act as a plasticizer, allowing chain mobility of the fibroin molecules to promote the formation of hydrogen bonds, leading to enhanced beta sheet secondary structure. This process is also referred to herein as "water vapor annealing."

Without wishing to be bound by theory, physical temperature-controlled water vapor annealing (TCWVA) is believed to provide a simple and effective method to obtain improved control of the molecular structure of silk biomaterials. Silk materials can be fabricated with controlled crystallinity from low beta-sheet content (α-helix predominant silk I structure) using conditions at 4°C to higher beta-sheet content (β-sheet predominant silk II structure) with about 60% crystallinity at 100°C. This physical approach encompasses the range of structures previously reported to dominate crystallization during the fabrication of silk materials, but provides a simpler, greener chemistry approach with precise control of reproducibility. Water annealing or water vapor annealing is described in, for example, PCT Application Nos. PCT/US2004/011199, filed April 12, 2004, and PCT/US2005/020844, filed June 13, 2005; and described in the literature [Jin et al., Adv. Funct. Mats. 2005, 15: 1241] and [Hu et al., Biomacromolecules, 2011, 12(5): 1686-1696], the contents of which are all incorporated herein by reference.

Another mode of annealing is by slow and controlled evaporation of water from the silk fibroin within the silk material/matrix. Slow and controlled drying is described, for example, in the literature [Lu et al., Acta. Biomater. 2010, 6(4): 1380-1387].

The annealing step can be performed for varying periods of time in a vapor environment, such as in a chamber filled with vapor. Without wishing to be bound by theory, the length of the annealing affects the amount of beta-sheet crystallinity obtained in the silk fibroin within the silk-based material. Thus, a typical annealing period can range from several seconds to several days. In some embodiments, the annealing is performed for a period of time from several seconds to several hours. For example, the annealing time can range from several seconds (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds) to about 2, 6, 12, 24, 36, or 48 hours.

The temperature of the steam used in the annealing process affects the amount of beta-sheet crystallinity obtained (see literature [HU et al., Biomacromolecules, 12: 1686-1696]). Therefore, the annealing can be performed at any desired temperature. For example, the annealing can be performed at a steam temperature of about 4°C to about 120°C. The optimal steam temperature for obtaining the required amount of beta-sheet crystallinity in silk fibroin among silk-based materials can be calculated according to the following equation (I).

C=a(1-exp(-kT)) (I)

(where C is the degree of beta-sheet crystallinity, a is 62.59, k is 0.028, and T is the annealing temperature; see reference [HU et al., Biomacromolecules, 12: 1686-1696]).

Without wishing to be bound by theory, the pressure at which the annealing takes place may also affect the degree or amount of beta-sheet crystallinity. In some embodiments, the contacting may be performed under a vacuum environment.

The relative humidity at which annealing occurs can also affect the degree or amount of beta-sheet crystallinity. The relative humidity at which the silk-based material is in contact with water or water vapor can range from about 5% to 100%. For example, the relative humidity can be from about 5% to about 95%, from about 10% to about 90%, or from about 15% to about 85%. In some embodiments, the relative humidity is greater than 90%.

Another useful method of annealing silk fibroin is to dehydrate the silk-based material using an organic solvent, such as an alcohol, e.g., methanol, ethanol, isopropyl, acetone, and the like. Such solvents have the effect of dehydrating the silk fibroin, which promotes "packing" of the silk fibroin molecules into a beta sheet structure. In some embodiments, the silk-based material can be treated with an alcohol, e.g., methanol, ethanol, and the like. The alcohol concentration can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. In some embodiments, the alcohol concentration is about 90%.

Thus, in some embodiments, the conformational change of the silk fibroin can be induced by immersion in an alcohol, such as methanol, ethanol, or the like. The alcohol concentration can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. In some embodiments, the alcohol concentration is 100%. When the conformational change is due to immersion in a solvent, the silk composition can be washed, for example, using a solvent/water gradient, to remove any residual solvent used in the immersion. The washing can be repeated, for example, once, twice, three times, four times, five times, or more times.

Alternatively, the conformational change of silk fibroin can be induced by shear stress. The shear stress can be applied, for example, by passing the silk composition through a needle. Other methods of inducing conformational changes include applying an electric field, applying pressure, or changing salt concentration.

The treatment time to induce the conformational change can be any period of time that provides the desired Silk II (beta-sheet crystallinity) content. In some embodiments, the treatment time can be in the range of about 1 hour to about 12 hours, about 1 hour to about 6 hours, about 1 hour to about 5 hours, about 1 hour to about 4 hours, or about 1 hour to about 3 hours. In some embodiments, the sintering time can be in the range of about 2 hours to about 4 hours or 2.5 hours to about 3.5 hours.

When the conformational change is induced by solvent immersion, the treatment time can range from several minutes to several hours. For example, the solvent immersion can be for a period of at least about 15 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 6 hours, at least about 18 hours, at least about 12 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days. In some embodiments, the solvent immersion can be for a period of from about 12 hours to about 7 days, from about 1 day to about 6 days, from about 2 to about 5 days, or from about 3 to about 4 days.

After treatment to induce a conformational change, the silk fibroin can comprise a silk II beta-sheet crystallinity content of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, but not 100% (i.e., all of the silk is in the silk II beta-sheet conformation). In some embodiments, the silk fibroin in the silk-based material comprises a beta-sheet crystallinity of at least 10%, for example, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 70%, 85%, 90%, 95%, but not 100% (i.e., not all of the silk fibroin is in the beta-sheet conformation). In some embodiments, the silk is entirely in the silk II beta-sheet conformation, i.e., 100% silk II beta-sheet crystallinity.

Regardless of the annealing method employed, the end result of the annealing process is often annealed silk fibroin having a high crystallinity that renders it insoluble. In some embodiments, "high crystallinity" refers to a beta sheet content of between about 20% and about 70%, for example, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, and about 75%.

In some embodiments, the annealing process can provide a silk-based material having a silk II beta-sheet crystallinity content of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, but not 100% (i.e., all of the silk is in the silk II beta-sheet conformation). In some embodiments, the silk-based material can have a silk II beta-sheet crystallinity of 100%.

In some embodiments, the silk fibroin in the provided silk fibroin composition has a protein structure that substantially comprises β-turn and β-strand regions. Without wishing to be bound by theory, it is believed that the silk β sheet content can affect the gel function and in vivo life of the composition. It should be understood that compositions comprising non-β sheet content (e.g., e-gels) may also be used. In aspects of these embodiments, the silk fibroin in the provided composition has a protein structure comprising, for example, about 5% β-turn and β-strand region, about 10% β-turn and β-strand region, about 20% β-turn and β-strand region, about 30% β-turn and β-strand region, about 40% β-turn and β-strand region, about 50% β-turn and β-strand region, about 60% β-turn and β-strand region, about 70% β-turn and β-strand region, about 80% β-turn and β-strand region, about 90% β-turn and β-strand region, or about 100% β-turn and β-strand region. In another aspect of the above embodiment, the silk fibroin in the low molecular weight silk fibroin composition has a protein structure comprising, for example, at least 10% β-turn and β-strand regions, at least 20% β-turn and β-strand regions, at least 30% β-turn and β-strand regions, at least 40% β-turn and β-strand regions, at least 50% β-turn and β-strand regions, at least 60% β-turn and β-strand regions, at least 70% β-turn and β-strand regions, at least 80% β-turn and β-strand regions, at least 90% β-turn and β-strand regions, or at least 95% β-turn and β-strand regions. In another aspect of these embodiments, the silk fibroin of the low molecular weight silk fibroin composition comprises, for example, about 10% to about 30% β-turn and β-strand region, about 20% to about 40% β-turn and β-strand region, about 30% to about 50% β-turn and β-strand region, about 40% to about 60% β-turn and β-strand region, about 50% to about 70% β-turn and β-strand region, about 60% to about 80% β-turn and β-strand region, about 70% to about 90% β-turn and β-strand region, about 80% to about 100% β-turn and β-strand region, about 10% to about 40% β-turn β-strand region, about 30% to about A protein structure comprising about 60% β-turn and β-strand region, about 50% to about 80% β-turn and β-strand region, about 70% to about 100% β-turn and β-strand region, about 40% to about 80% β-turn and β-strand region, about 50% to about 90% β-turn and β-strand region, about 60% to about 100% β-turn and β-strand region, or about 50% to about 100% β-turn and β-strand region. In some embodiments, a silk β sheet content of less than 10% to about 55% can be used in the silk fibroin compositions disclosed herein.

In some embodiments, the silk fibroin of the silk fibroin compositions provided herein has a protein structure substantially free of α-helix and random coil regions. In aspects of these embodiments, the silk fibroin has a protein structure comprising, for example, about 5% α-helix and random coil regions, about 10% α-helix and random coil regions, about 15% α-helix and random coil regions, about 20% α-helix and random coil regions, about 25% α-helix and random coil regions, about 30% α-helix and random coil regions, about 35% α-helix and random coil regions, about 40% α-helix and random coil regions, about 45% α-helix and random coil regions, or about 50% α-helix and random coil regions. In other aspects of these embodiments, the silk fibroin has a protein structure comprising, for example, at most 5% α-helix and random coil regions, at most 10% α-helix and random coil regions, at most 15% α-helix and random coil regions, at most 20% α-helix and random coil regions, at most 25% α-helix and random coil regions, at most 30% α-helix and random coil regions, at most 35% α-helix and random coil regions, at most 40% α-helix and random coil regions, at most 45% α-helix and random coil regions, or at most 50% α-helix and random coil regions. In another aspect of these embodiments, the silk fibroin comprises, for example, about 5% to about 10% α-helix and random coil region, about 5% to about 15% α-helix and random coil region, about 5% to about 20% α-helix and random coil region, about 5% to about 25% α-helix and random coil region, about 5% to about 30% α-helix and random coil region, about 5% to about 40% α-helix and random coil region, about 5% to about 50% α-helix and random coil region, about 10% to about 20% α-helix and random coil region, about 10% to about 30% α-helix and random coil region, about 15% to about 25% α-helix and random coil region, about 15% to about 30% α-helix and random coil region, or about It has a protein structure comprising 15% to about 35% α-helix and random coil regions.

In another aspect, the present disclosure relates to methods of altering one or more properties of a silk fibroin article or article of manufacture described herein. Generally, the methods comprise varying the weight ratio of various silk fibroin fragments among the low molecular weight silks in the silk fibroin article or article of manufacture. In some embodiments, the methods comprise varying the weight ratio of silk fibroin fragments having a molecular weight within a first predetermined range of about 3.5 kDa to about 120 kDa to silk fibroin fragments having a molecular weight within a second predetermined range of about 3.5 kDa to about 120 kDa, wherein the first and second predetermined ranges do not overlap. In some embodiments, the methods comprise varying the weight ratio of silk fibroin fragments having a molecular weight greater than 200 kDa to silk fibroin fragments having a molecular weight within a predetermined range of about 3.5 kDa to about 120 kDa.

Without limitation, the properties that can be modified using the methods described herein can be selected from the group consisting of release rate of the formulation present in the article, release kinetics of the formulation present in the article, resolubility, degradation, mechanical properties, optical properties, porosity, pore size, viscosity, biocompatibility, bioresorbability, net charge of the article, particle size, and any combination thereof.

In another aspect, the present disclosure provides a method of controlling the size of silk particles. The method generally comprises varying the weight ratio of various silk fibroin fragments of low molecular weight silk in a solution and forming silk particles from the solution. In some embodiments, the method comprises varying the weight ratio of silk fibroin fragments having a molecular weight within a first predetermined range of about 3.5 kDa to about 120 kDa to silk fibroin fragments having a molecular weight within a second predetermined range of about 3.5 kDa to about 120 kDa, wherein the first and second predetermined ranges do not overlap. In some embodiments, the method comprises varying the weight ratio of silk fibroin fragments having a molecular weight greater than 200 kDa to silk fibroin fragments having a weight ratio within a predetermined range of about 3.5 kDa to about 120 kDa. Methods of making silk particles are described, for example, in International Application WO2011/041395; International Publication No. WO2008/118133; U.S. Patent Application Publication No. US2010/0028451; U.S. Provisional Application Serial No. 61/719,146 (filed October 26, 2012); and Wenk et al. J Control Release, 2008; 132: 26-34, the contents of which are all incorporated herein by reference.

In all embodiments described herein, the low molecular weight silk fibroin fragments can be derived from a portion or portions of a full-length silk fibroin polypeptide, such that the amino acid sequence of the low molecular weight silk fibroin collectively represents less than 100% of the full-length silk fibroin polypeptide. For example, one or more recombinantly produced silk fibroin polypeptides can be used to perform some embodiments of any aspect described herein. Such recombinant silk fibroin polypeptides can contain a fragment or fragments of the full-length counterpart. In some embodiments, a silk fibroin polypeptide corresponding to a fragment or fragments of the full-length counterpart can be produced from a transgenic organism carrying the transgene to produce such a fragment.

In any of the embodiments described herein, the low molecular weight silk fibroin fragments can comprise one or more mutations and/or modifications relative to a naturally occurring (e.g., wild-type) sequence of one or more silk fibroin fragments. Such mutations and/or modifications of the silk fibroin fragments can be naturally occurring or introduced by design. For example, in some embodiments, such mutations and/or modifications in the silk fibroin fragments can be introduced using recombinant techniques, chemical modifications, or the like.

Silk fibroin is an example of a polypeptide having a portion or portions of an amino acid sequence capable of assuming a beta-sheet secondary structure. For example, the silk fibroin structure generally comprises a sequence of amino acids in which the portion or portions of the sequence generally feature alternating glycines and alanines or alanines alone. Without wishing to be bound by theory, it is believed that this arrangement allows the fibroin molecules to self-assemble into a beta-sheet conformation. Thus, in another aspect, provided herein is a composition comprising a population of polypeptide fragments having a portion or portions of an amino acid sequence in which the portion or portions of the sequence feature alternating glycines and alanines or alanines alone and having a molecular weight ranging from about 3.5 kDa to about 120 kDa or from about 5 kDa to about 125 kDa. In some embodiments, the composition is characterized in that no more than 15% of the total number of polypeptide fragments in the population have a molecular weight greater than 200 kDa, and more than 50% of the total number of polypeptide fragments in the population have a molecular weight within a range of about 3.5 kDa to about 120 kDa, or about 5 kDa to about 125 kDa.

In another aspect, the silk fibroin in the compositions and/or methods described herein may be replaced with or used in combination with other non-silk polypeptides that comprise a beta sheet structure or have a tendency to form such a structure based on their amino acid sequence. Accordingly, also provided herein are polypeptide compositions comprising a population of beta-sheet forming polypeptide fragments. In some embodiments, the population of beta-sheet forming polypeptide fragments described herein can have a wide range of molecular weights, with no more than 15% of the total number of beta-sheet forming polypeptide fragments in the population having a molecular weight greater than 200 kDa, and greater than 50% of the total number of beta-sheet forming polypeptide fragments in the population having a molecular weight within a predetermined range of from about 3.5 kDa to about 120 kDa or from about 5 kDa to about 125 kDa.

The term "beta-sheet forming polypeptide" as used herein refers to a polypeptide having a portion or portions of an amino acid sequence that assumes a beta-sheet secondary structure. In some embodiments, a beta-sheet forming polypeptide can be selected based on its tendency to form such a structure based on its beta sheet structure or amino acid sequence.

In some embodiments, the beta-sheet forming polypeptide can have amphiphilic properties (i.e., having both hydrophilic and hydrophobic portions). The amphiphilic polypeptide can be obtained from a single source (e.g., a naturally occurring protein) and has both a hydrophobic module or stretch and a hydrophilic module or stretch within the polypeptide, such that the single polypeptide itself is inherently amphiphilic. In some embodiments, the hydrophobic module or stretch and the hydrophilic module or stretch can be fused or coupled to each other to form an amphiphilic entity. Such "fusion" or "chimeric" polypeptides can be produced using recombinant techniques, chemical coupling, or both.

In some embodiments, the beta-sheet forming polypeptide can comprise a portion or portions of the amino acid sequence of a polypeptide selected from the following list: fibroin, actin, collagen, catenin, chitosan, claudin, choline, elastin, elaunin, extensin, fibrillin, lamin, laminin, keratin, tubulin, viral structural protein, zein protein (seed storage protein) and any combination thereof.

In some embodiments, the beta-sheet forming polypeptide can comprise a regenerated (e.g., purified) protein from a natural source, a recombinant protein produced in a heterologous system, a synthetic or chemically produced peptide, or a combination thereof.

In some embodiments, the beta-sheet forming polypeptide can comprise a portion or portions of the amino acid sequence of a polypeptide corresponding to any one of the lists provided above, and may or may not have one or more sequence variants relative to the native or wild-type counterpart. For example, in some embodiments, such variants can exhibit at least 85% overall sequence identity relative to the wild-type sequence, for example, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% overall sequence identity.

Specific exemplary manufacturing methods

Without wishing to be bound by theory, it is believed that many of the problems associated with forming articles comprising silk fibroin can be reduced or limited by treating them with heat or pressure prior to processing them into their final desired shape. This provides the ability to fabricate articles having consistent geometries without significant bubbles, shrinkage, and distortion. This provides reproducible, consistent samples for testing and implantation. In addition, the articles can be machined, which allows for precise replication of biological geometries, for example, by computer numerical control (CNC) milling of the silk blank into the desired structure. Methods including treating silk fibroin articles with heat or high pressure prior to processing them into their final desired configuration are described, for example, in U.S. Provisional Application Nos. 61/808,768 (filed Apr. 5, 2013), 61/719,146 (filed Oct. 26, 2012), 61/809,535 (filed Apr. 8, 2013), and 61/881,653 (filed Sep. 24, 2013), the contents of all of which are incorporated herein by reference in their entireties.

Accordingly, in one aspect, the present disclosure provides a method of making a silk fibroin article. Generally, the method comprises incubating a silk fibroin composition (e.g., a low molecular weight silk fibroin composition) at an elevated temperature and/or under an elevated pressure. The silk fibroin in the composition can be at least partially insoluble. After the incubation, the composition can be processed into a desired final shape. Without limitation, the silk fibroin composition for making the article can be in the form of a solution, a slurry, a suspension, a colloid, a mixture, a dispersion, a paste, or the like.

The term "insoluble state" as used herein, when used with respect to silk fibroin, refers to a substantially amorphous, primary beta-sheet conformational configuration or state. The term "formed into an insoluble state" is not intended to refer to polymerization of silk monomers into silk polymers. Rather, it is intended to refer to conversion of soluble silk fibroin into a water-soluble state. As used herein, silk fibroin is "insoluble" if the silk fibroin can be pelleted by centrifugation or does not dissolve upon immersion in water or washing with water at 37° C. or less.

In some embodiments, the silk fibroin composition can include an organic solvent. In one embodiment, the organic solvent can be hexafluoroisopropanol (HFIP). In some other embodiments, the silk fibroin composition is free or essentially free of an organic solvent, i.e., free of solvents other than water. Without wishing to be bound by theory, it is believed that the organic solvent in the silk fibroin composition allows for more uniform drying of the composition, resulting in more uniform mechanical and structural properties in the final silk fibroin manufactured article. In addition, the use of an organic solvent also provides better mechanical and structural properties for processing into a final shape.

In some embodiments, the method comprises: (i) providing silk fibroin, wherein the silk fibroin is at least partially insoluble; (ii) incubating the composition at elevated temperature and/or elevated pressure; (iii) optionally repeating step (ii); and (iv) processing the composition into a desired shape. The term "incubating" as used herein means subjecting the composition to elevated temperature and/or elevated pressure.

In some embodiments, the silk fibroin composition is within a mold. The term "mold" as used herein is intended to include any mold, vessel, or substrate capable of forming, holding, or supporting the silk fibroin composition. Thus, the simplest form of the mold may merely include a support surface. The mold may be of any desired shape and may be made of any suitable material, including polymers (e.g., polysulfone, polypropylene, polyethylene), metals (e.g., stainless steel, titanium, cobalt chromium), ceramics (e.g., alumina, zirconia), glass ceramics, and glasses (e.g., borosilicate glasses). In some embodiments, the mold may provide a scaffold of simple geometry that may be processed into a final desired shape, i.e., the mold may be used to provide a blank that may be processed into a final shape.

Thus, in some embodiments, the method comprises: (i) providing a mold comprising a silk fibroin composition wherein the silk fibroin in the composition is at least partially insoluble; (ii) incubating the composition at elevated temperature or under pressure; (iii) optionally repeating step (ii) one or more times; and (iv) processing the composition into a desired shape.

In some embodiments, the step of providing a mold comprising a silk fibroin composition comprises transferring a solution comprising silk fibroin to the mold to induce a conformational change in the silk fibroin.

The term "elevated temperature" as used herein means a temperature greater than room temperature. Typically, the elevated temperature is a temperature greater than about 25° C. For example, the elevated temperature can be at least about 30° C., at least about 35° C., at least about 40° C., at least about 45° C., at least about 50° C., at least about 55° C., at least about 60° C., at least about 65° C., at least about 70° C., at least about 75° C., at least about 80° C., at least about 85° C., at least about 90° C., at least about 95° C., at least about 100° C., at least about 105° C., at least about 110° C., at least about 115° C., at least about 120° C., at least about 125° C., at least about 130° C., at least about 135° C., at least about 140° C., at least about 145° C., or at least about 150° C. In some embodiments, the elevated temperature is at least about 121° C.

The term "pressure" as used herein means a pressure of greater than or equal to about 0.05 bar, about 0.1 bar, about 0.15 bar, about 0.2 bar, about 0.25 bar, about 0.3 bar, about 0.35 bar, about 0.4 bar, about 0.45 bar, about 0.5 bar, about 0.55 bar, about 0.6 bar, about 0.65 bar, about 0.7 bar, about 0.75 bar. For example, the pressure can be about 1 bar, 1.25 bar, 1.5 bar, 1.75 bar, 2 bar, 2.25 bar, 2.5 bar, 2.75 bar, 3 bar, 3.25 bar, 3.5 bar, 3.75 bar, 4 bar, 4.25 bar, 4.5 bar, 4.75 bar, 5 bar, 5.25 bar, 5.5 bar, 5.75 bar, 6 bar, 7.25 bar, 7.5 bar, 7.75 bar, 8 bar, 8.25 bar, 8.5 bar, 8.75 bar, 9 bar, 9.25 bar, 9.5 bar, 9.75 bar, 10 bar, or more. In some embodiments, the pressure is about 1 bar or more. In some embodiments, the incubation is under vacuum.

In some embodiments, the incubation is in the presence of water vapor.

Without limitation, the incubation can be for any desired period of time. For example, the incubation can be for a period of about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 8 minutes, about 10 minutes or more. In some embodiments, the incubation can be for a period of about 10 minutes to about 5 hours, about 15 minutes to about 2.5 hours, about 20 minutes to about 2 hours, about 25 minutes to about 1.5 hours. In some embodiments, the incubation is for about 25 minutes.

The properties of a number of manufactured articles (e.g., strength, molecular weight, degradation profile, swelling properties, density, color, etc.) can be controlled by repeating the incubation step. Thus, in some embodiments, the incubation step can be repeated, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more times.

In some embodiments, the silk fibroin composition can optionally be dried prior to repeating the incubation step. Without limitation, the drying can be for any desired period of time. For example, the drying can be for a period of about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, or more. In some embodiments, the incubation can be for a period of about 5 minutes to about 5 hours, about 10 minutes to about 2.5 hours, about 15 minutes to about 2 hours, about 20 minutes to about 1.5 hours. In some embodiments, the drying can be for about 15 minutes. Additionally, the drying can be at room temperature or at an elevated temperature. For example, such drying can be at a temperature of about 4°C to about 100°C, about 10°C to about 95°C, about 15°C to about 90°C, about 20°C to about 85°C, about 25°C to about 80°C, about 30°C to about 75°C, about 35°C to about 60°C, or about 45°C to about 65°C.

In some embodiments, the incubation comprises autoclaving. "Autoclaving" means heat treatment in the presence or absence of steam (e.g., saturated steam) at superatmospheric pressure. For the purposes of the present disclosure, the term autoclaving refers to the process of raising the temperature of a composition under sufficient pressure for a sufficient period of time. In general, autoclaving relates to a standardized thermal heating procedure characterized by the following parameters: heating under pressure to a temperature of at least 120° C. for a period of at least 15 minutes. The autoclaving temperature can be in the range of 120 to 150° C., more preferably 120 to 140° C.; and the pressure can be in the range of 1 to 20 bar, more preferably 1 to 10 bar, more preferably 1 to 5 bar. The time required can be in the range of 15 to 120 minutes, more preferably 15 to 60 minutes. In some embodiments, the autoclaving can be repeated, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more.

Without wishing to be bound by theory, it is thought that steam, high heat, and/or pressure forces water into the sample during autoclaving, where the water acts as a plasticizer, allowing the chains to be mobile. During the drying cycle, the mobile chains are compacted, producing a denser, stronger material. Over time and with the application of heat, the protein chains may be broken down. This may allow for a tighter packing, avoiding some of the chain entanglement. Thus, changes in the parameters of the autoclaving process, such as slow or fast venting, temperature, pressure, and duration, may affect the mechanical, physical, or structural properties of the resulting material.

In some embodiments, the silk fibroin composition can optionally be dried after incubation, but prior to processing into a desired shape or geometry. Without limitation, drying prior to processing into a desired shape can be for any desired period of time. For example, drying can be for a period of from about several minutes to about several days. In some embodiments, drying can be for a period of at least about 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. In some embodiments, drying prior to processing into said final shape can be for at least about 7 days. Additionally, said drying can be at room temperature or at an elevated temperature. For example, such drying can be at a temperature of about 4° C. to about 100° C., about 10° C. to about 95° C., about 15° C. to about 90° C., about 20° C. to about 85° C., about 25° C. to about 80° C., about 30° C. to about 75° C., about 35° C. to about 60° C., or about 45° C. to about 65° C. In some embodiments, the drying is at a temperature of about 60° C.

After optionally drying the silk fibroin composition, the composition may be processed into a final desired shape. The term "processing" as used herein with respect to processing into a desired shape should be understood to include any method or process used to provide a manufactured article of a final shape. Without limitation, such processing may include, but is not limited to, mechanical and chemical means. For example, processing may be selected from the group consisting of machining, turning, rolling, thread rolling, drilling, milling, sanding, punching, die cutting, blanking, broaching, extruding, chemical etching, and any combination thereof. The term "machining" as used herein should be understood to include all types of machining operations, including but not limited to CNC machining, cutting, milling, turning, drilling, forming, planing, broaching, sawing, burnishing, grinding, and the like. More complex and intricate geometries may be achieved by using a combination of more than one processing method. The term "machinable" means that a material can be readily machined.

After being processed into a desired shape, the manufactured article may be further post-treated. For example, the manufactured article may be incubated at elevated temperature or under pressure. Post-treatment of the article may be used to control a number of properties of the manufactured article, such as strength, molecular weight, degradation profile, swelling, density, color, etc. In some embodiments, the post-treatment comprises autoclaving the article into its final shape one or more times, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times.

As discussed above, the silk fibroin composition may optionally be dried after the incubating step. Thus, in some embodiments, the method comprises the steps of: (i) providing a mold comprising a silk fibroin composition, wherein the silk fibroin in the composition is at least partially insoluble; (ii) incubating the composition at an elevated temperature or under pressure; (iii) drying the composition; (iv) optionally repeating steps (ii) and (iii) one or more times; and (iv) processing the composition into a desired shape.

The present disclosure also provides silk fibroin articles prepared by the methods disclosed herein. Without limitation, the articles may be used in medical applications, such as medical devices, or the articles may be used in non-medical applications. As used herein, the term "medical device" is intended to encompass all types of medical devices, including those used in conjunction with external or internal treatment of a mammal. Medical devices used in external treatment of a mammal include, but are not limited to, wound dressings, burn dressings or other skin coverings, and surgical sutures. Medical devices used in internal treatment of a mammal include, but are not limited to, vascular grafts, stents, catheters, valves, artificial joints, artificial organs, surgical sutures, and the like.

Exemplary medical devices include, but are not limited to, orthopedic implants, facial implants, nasal implants (e.g., nasal reconstructions), suture anchors, dental implants, Swanson prostheses, and any combination thereof. In some embodiments, the article of manufacture is a continuous one-phase suture anchor.

As used herein, the term "orthopedic implant" includes within its scope any device for implantation into the body of a vertebrate, particularly a mammal, such as a human, for the preservation or restoration of function of the musculoskeletal system, particularly joints and bones, and for the relief of pain in these structures. Exemplary orthopedic implants include, but are not limited to, an orthopedic screw, an orthopedic plate, an orthopedic rod, an orthopedic tulip, or any combination thereof.

In some embodiments, the article of manufacture can be a tapping screw, for example, a self-tapping screw.

In some embodiments, the article of manufacture may be a suture anchor. The suture anchor comprises an anchor, an eyelet, and a suture. The anchor is inserted into the bone and may be a screw mechanism or an interference fit, and the eyelet is a hole or loop in the anchor through which the suture passes.

In some embodiments, the article of manufacture may be a dental implant. As used herein, the term "dental implant" includes within its scope any device for implantation into the oral cavity of a vertebrate, particularly a mammal, such as a human, in a dental restoration procedure. A dental implant may also be referred to as a dental prosthetic device. Typically, a dental implant is comprised of one or more implant parts. For example, a dental implant typically includes a second implant part, such as an abutment, and/or a dental fixture coupled to a dental restoration, such as a crown, bridge, or denture. However, any device for implantation, such as a dental fixture, may be referred to as an implant alone, even when other parts are connected thereto. A dental implant is presently a preferred embodiment.

In some embodiments, the dental implant consists of a crown and a screw implant. The implant is inserted into the bone using a screw mechanism to stabilize the crown.

*In some embodiments, the article of manufacture may be a bone screw and/or a bone plate. A bone screw comprises a threaded portion and an associated device, such as a head, that is used to insert and stabilize the bone plate.

In some embodiments, the article of manufacture may be a Swanson prosthesis. The Swanson Finger Joint Implant is a flexible, intramedullary-stemmed, 1-piece implant that aids in restoring hand and wrist function impaired by rheumatoid, degenerative, or traumatic arthritis. It is composed of a silicone elastomer, and its primary function is to aid in maintaining adequate joint space and alignment with excellent lateral stability and minimal flexion-extension motion limitation. These implants are subject to minimal loads because most of the compressive load is distributed to the bone.

In some embodiments, the article of manufacture may be used for nasal reconstruction. Nasal reconstruction is performed to create a nose that is functional but not aesthetically unsightly. Structural grafts are often needed to provide rigidity to the lateral wall, resist lateral collapse, and establish nasal contour and projection. Conventional materials include alloplasts, such as silicone and porous high-density polyethylene, as well as allografts, such as alloderm or costal cartilage.

The methods disclosed herein enable silk to be used in such applications because of its ability to be formed into a variety of different shapes. For example, the silk can be extruded into a rod and then cut (lathe) to remove any distortion and achieve the desired size. Additionally, the structure can be formed in-situ to conform to the unique geometry of the nose.

In some embodiments, the article of manufacture may be used in otoplasty. Otoplasty is a procedure to reconstruct partial or complete ear defects, typically resulting from congenital underdevelopment, trauma, cancer resection, and protruding ears. The ear may be reconstructed using cartilage from the rib cage, or an artificial ear may be created. Costal cartilage is carved and joined together using fine stainless steel wires to create a very delicate framework.

The methods disclosed herein allow for extruding, drawing, and forming silk in situ into a network of fine wires that can be used to create a framework for a new ear. Furthermore, because of the numerous processes that can create complex geometries, the silk can be manufactured based on the specific needs of the patient.

In addition to the specific medical devices and implants discussed above, the methods disclosed herein can be used for facial implants (dermal fillers, cheek implants, orbitals), occuloplasty, lip enhancement, reproductive plastic surgery (penile implants, vaginaplasty, sex reassignment), buttock augmentation, and other soft tissue "plasty."

Non-medical applications include the manufacture of dice, pins, bullets, children's toys (e.g., building blocks, Legos, Checkers, etc.), and biodegradable plastic substitutes.

Additives

In some embodiments, the silk fibroin compositions described herein can include at least one additive (e.g., replacing or in addition to an active agent/sample/component discussed herein; or in some embodiments the active agent/sample/component may also be an “additive” as that term is used herein). In some embodiments of the various aspects described herein, the silk composition can further include one or more (e.g., 1, 2, 3, 4, 5, or more) additives. Without wishing to be bound by theory, it is believed that the additives can provide one or more desirable properties, such as strength, flexibility, ease of processing and handling, biocompatibility, bioresorability, surface morphology, release rate, and/or kinetics of the one or more active agents present in the composition. The additives can be covalently or non-covalently linked to the silk fibroin and can be uniformly or non-uniformly present within the silk composition.

For example, a silk material can be prepared from a fibroin solution comprising one or more (e.g., 1, 2, 3, 4, 5 or more) additives.

Without limitation, the additive can be selected from small organic or inorganic molecules; saccharides; oligosaccharides; polysaccharides; polymers; proteins; peptides; peptide analogs and derivatives; peptidomimetics; nucleic acids; nucleic acid analogs; and the like. In some embodiments, the additive is or comprises an immunogen; an antigen; an extract prepared from a biological material, such as a bacterial, plant, fungal, or animal cell; an animal tissue; a natural or synthetic composition; and any combination thereof. Additionally, the additive can be in any physical form. For example, the additive can be in the form of a particle, a fiber, a film, a gel, a mesh, a mat, a non-woven mat, a powder, a liquid, or any combination thereof. In some embodiments, the additive is a particle.

In one embodiment, the at least one additive is a stabilizing agent that can be used to stabilize one or more of the composition, and/or the active agent/sample/component included in the composition, and/or to increase the solubility of the silk fibroin. One example of a stabilizing agent that can be added to stabilize at least RNA or protein present in the biological sample includes a nuclease inhibitor (e.g., an RNAse inhibitor) or a proteinase inhibitor, respectively.

Additional examples of stabilizers include, but are not limited to, amino acids such as sodium glutamate, arginine, lysine, and cysteine; monosaccharides such as glucose, galactose, fructose, and mannose; disaccharides such as sucrose, maltose, and lactose; sugar alcohols such as sorbitol and mannitol; polysaccharides such as oligosaccharides, starch, cellulose, and derivatives thereof; human serum albumin and bovine serum albumin; gelatin, and gelatin derivatives such as hydrolyzed gelatin; and the antioxidant ascorbic acid; and ions (e.g., cationic or anionic stabilizers); and surfactants; and any combination thereof. These materials are described, but are not limited to, in publications such as "Toketsu-Kanso To Hogo Busshitsu (Lyophilization And Protective Materials)" by Nei, p. 1-176, published by Tokyo Daigaku Shuppan Kai (Publishing Association of the University of Tokyo), Japan, 1972; and "Shinku Gijutsu Koza (8): Sinku Kanso (Lecture on Vacuum Technology (8): Vacuum Drying)" Ota et al., p. 176-182, published by Nikkan Kogyo Shimbun Co., Ltd., Japan, 1964.

In some embodiments, the additive is a biocompatible polymer. Exemplary biocompatible polymers include, but are not limited to, polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polyesters, poly(ortho esters), poly(phosphazines), poly(phosphate esters), polycaprolactone, gelatin, collagen, fibronectin, keratin, polyaspartic acid, alginates, chitosan, chitin, hyaluronic acid, pectins, polyhydroxyalkanoates, dextrans, and polyanhydrides, polyethylene oxide (PEO), poly(ethylene glycol) (PEG), triblock copolymers, polylysine, alginates, polyaspartic acid, any derivatives thereof, and any combinations thereof. Other exemplary biocompatible polymers suitable for use according to the present disclosure include, but are not limited to, those described in, for example, US Pat. Nos. 6,302,848; 6,395,734; Nos. 6,127,143; 5,263,992; 6,379,690; 5,015,476; 4,806,355; 6,372,244; 6,310,188; 5,093,489; US 387,413; 6,325,810; 6,337,198; US 6,267,776; 5,576,881; 6,245,537; 5,902,800; and 5,270,419, the entire contents of which are incorporated herein by reference.

In one embodiment, the additive is glycerol, which can affect the flexibility and/or solubility of the silk-based material. Silk-based materials, such as silk films comprising glycerol, are described in WO 2010/042798, the contents of which are incorporated herein by reference in their entirety.

Examples of other additives include, but are not limited to, cell adhesion mediators known to affect cell adhesion, such as collagen, elastin, fibronectin, vitronectin, laminin, proteoglycans, or peptides containing known integrin binding domains, e.g., the "RGD" integrin binding sequence, or modifications thereof (Schaffner P & Dard 2003 Cell Mol Life Sci. Jan;60(1):119-32; Hersel U. et al. 2003 Biomaterials. Nov;24(24):4385-415); biologically active ligands; and agents that enhance or inhibit specific modifications of cell or tissue ingrowth. Other examples of additives that enhance proliferation or differentiation include, but are not limited to, osteoinductive agents, such as bone morphogenetic proteins (BMPs); Cytokines, growth factors including, but not limited to, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factors (IGF-I and II), TGF-β1, etc.

The total amount of additives in the composition can be from about 0.01 wt % to about 99 wt %, from about 0.01 wt % to about 70 wt %, from about 5 wt % to about 60 wt %, from about 10 wt % to about 50 wt %, from about 15 wt % to about 45 wt %, or from about 20 wt % to about 40 wt % of the total silk composition. In some embodiments, the ratio of silk fibroin to additive in the composition can range from about 1000:1 (w/w) to about 1:1000 (w/w), from about 500:1 (w/w) to about 1:500 (w/w), from about 250:1 (w/w) to about 1:250 (w/w), from about 200:1 (w/w) to about 1:200 (w/w), from about 25:1 (w/w) to about 1:25 (w/w), from about 20:1 (w/w) to about 1:20 (w/w), from about 10:1 (w/w) to about 1:10 (w/w), or from about 5:1 (w/w) to about 1:5 (w/w).

In some embodiments, the total amount of additive in the silk-based material can be from about 0.1 wt % to about 70 wt %, from about 5 wt % to about 60 wt %, from about 10 wt % to about 50 wt %, from about 15 wt % to about 45 wt %, or from about 20 wt % to about 40 wt % of the total silk fibroin in the silk-based material. The skilled person can determine the appropriate ratio of silk fibroin to additive by, for example, measuring the properties of the component or silk-based material affected by adding the additive in various ratios as described herein.

In some embodiments, the composition has a molar ratio of silk fibroin to additive of, for example, at least 1000:1, at least 900:1, at least 800:1, at least 700:1, at least 600:1, at least 500:1, at least 400:1, at least 300:1, at least 200:1, at least 100:1, at least 90:1, at least 80:1, at least 70:1, at least 60:1, at least 50:1, at least 40:1, at least 30:1, at least 20:1, at least 10:1, at least 7:1, at least 5:1, at least 3:1, at least 1:1, at least 1:3, at least 1:5, at least 1:7, at least 1:10, at least 1:20, at least 1:30, at least 1:40, at least 1:50, at least 1:60, at least 1:70, at least 1:80, at least 1:90, at least 1:100, at least 1:200, at least 1:300, at least 1:400, at least 1:500, at least 1:600, at least 1:700, at least 1:800, at least 1:900, or at least 1:100.

In some embodiments, the composition has a molar ratio of silk fibroin to additive of, for example, at most 1000:1, at most 900:1, at most 800:1, at most 700:1, at most 600:1, at most 500:1, at most 400:1, at most 300:1, at most 200:1, 100:1, at most 90:1, at most 80:1, at most 70:1, at most 60:1, at most 50:1, at most 40:1, at most 30:1, at most 20:1, at most 10:1, at most 7:1, at most 5:1, at most 3:1, at most 1:1, at most 1:3, at most 1:5, at most 1:7, at most 1:10, at most 1:20, at most 1:30, at most 1:40, at most 1:50, at most 1:60, up to 1:70, up to 1:80, up to 1:90, up to 1:100, up to 1:200, up to 1:300, up to 1:400, up to 1:500, up to 1:600, up to 1:700, up to 1:800, up to 1:900, or up to 1:1000.

In some embodiments, the composition comprises a molar ratio of silk fibroin to additive of, for example, about 1000:1 to about 1:1000, about 900:1 to about 1:900, about 800:1 to about 1:800, about 700:1 to about 1:700, about 600:1 to about 1:600, about 500:1 to about 1:500, about 400:1 to about 1:400, about 300:1 to about 1:300, about 200:1 to about 1:200, about 100:1 to about 1:100, about 90:1 to about 1:90, about 80:1 to about 1:80, about 70:1 to about 1:70, about 60:1 to about 1:60, about 50:1 to about About 1:50, about 40:1 to about 1:40, about 30:1 to about 1:30, about 20:1 to about 1:20, about 10:1 to about 1:10, about 7:1 to about 1:7, about 5:1 to about 1:5, about 3:1 to about 1:3, or about 1:1.

In some embodiments, the additive is a biologically active agent. The term "biologically active agent" as used herein refers to any molecule that exerts one or more biological effects in the body. For example, a biologically active agent can be a therapeutic agent for treating or preventing a disease state or condition in a subject. Biologically active agents include, without limitation, organic molecules, inorganic substances, proteins, peptides, nucleic acids (e.g., genes, gene fragments, gene regulatory sequences, and antisense molecules), nucleoproteins, polysaccharides, glycoproteins, and lipoproteins. The types of biologically active compounds that can be included in the compositions described herein include, without limitation, anticancer agents, antibiotics, analgesics, anti-inflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anticonvulsants, hormones, muscle relaxants, sedatives, eye drops, prostaglandins, antidepressants, anti-psychotics, nutritional factors, osteogenic proteins, growth factors, and vaccines.

In some embodiments, the additive is a therapeutic agent. The term "therapeutic agent" as used herein means a molecule, group of molecules, complex, or substance that is administered to an organism for diagnostic, therapeutic, prophylactic, or veterinary purposes. The term "therapeutic agent" as used herein includes "drug" or "vaccine." The term includes topical, topical, and systemic pharmaceuticals, therapeutics, treatments, nutraceuticals, cosmeceuticals, biologics, devices, diagnostics, and contraceptives for human and animal use, both externally and internally, including agents useful for clinical and veterinary screening, prevention, prophylaxis, healing, wellness, detection, imaging, diagnostics, therapy, surgery, monitoring, cosmetics, prosthodontics, forensics, and the like. The term may also be used in reference to agropharmaceutical, occupational, military, industrial, and environmental therapeutics or treatments that include selected molecules or selected nucleic acid sequences capable of recognizing selected targets, including or capable of contacting cell receptors, membrane receptors, hormone receptors, therapeutic receptors, microorganisms, viruses, or plants, animals, and/or humans. The term also specifically encompasses therapeutically effective nucleic acids and compounds comprising nucleic acids, such as deoxyribonucleic acids (DNA), ribonucleic acids (RNA), nucleic acid analogs (e.g., locked nucleic acids (LNA), peptide nucleic acids (PNA), xeno nucleic acids (XNA)), or mixtures or combinations thereof, including DNA nanoplexes, siRNA, microRNA, shRNA, aptamers, ribozymes, decoy nucleic acids, antisense nucleic acids, RNA activators, and the like. In general, any therapeutic agent may be included in the compositions described herein.

The term "therapeutic agent" also includes agents that can provide a local or systemic biological, physiological, or therapeutic effect to the biological system to which it is applied. For example, a therapeutic agent may have, among other functions, control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, analgesic action, promote anti-cell adhesion, and enhance bone growth. Other suitable therapeutic agents may include antiviral agents, hormones, antibodies, or therapeutic proteins. Other therapeutic agents include prodrugs, which are agents that are not biologically active when administered, but are converted to a biologically active agent through metabolism or some other mechanism when administered to a subject. Additionally, the silk-based drug delivery composition may contain one therapeutic agent or a combination of two or more therapeutic agents.

Therapeutic agents can include a wide variety of different compounds, including compounds and mixtures of compounds, such as small organic or inorganic molecules; saccharins; oligosaccharides; polysaccharides; biological macromolecules, such as peptides, proteins, and peptide analogs and derivatives; peptide mimetics; antibodies and antigen-binding fragments thereof; nucleic acids; nucleic acid analogs and derivatives; biological materials, such as extracts prepared from bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combination thereof. In some embodiments, the therapeutic agent is a small molecule.

The term "small molecule" as used herein may refer to a "natural product-like" compound, however, the term "small molecule" is not limited to "natural product-like" compounds. Rather, small molecules are typically characterized by containing a few carbon-carbon bonds and having a molecular weight less than 5000 daltons (5 kDa), preferably less than 3 kDa, even more preferably less than 2 kDa, and most preferably less than 1 kDa. In some cases, small molecules having a molecular weight of less than 700 daltons are preferred.

Exemplary therapeutic agents include, but are not limited to, those found in the literature [Harrison's Principles of Internal Medicine, ^13th Edition, Eds. TR Harrison et al. McGraw-Hill NY, NY; Physicians' Desk Reference, ^50th Edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, ^8th Edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, USP XII NF XVII, 1990], the entire contents of which are incorporated herein by reference.

Therapeutics encompass the categories and specific examples disclosed herein. The categories are not intended to be limited to specific examples. Furthermore, those of ordinary skill in the art will recognize many other compounds that fall within the categories and are useful according to the present disclosure. Examples include radiosensitizers, steroids, xanthines, beta-2-agonist bronchodilators, anti-inflammatory agents, analgesics, calcium channel blockers, angiotensin-converting enzyme inhibitors, beta-blockers, centrally active alpha-agonists, alpha-1-antagonists, anticholinergics/spasmodics, vasopressin analogs, antiarrhythmics, antiparkinsonian agents, antianginals/antihypertensives, anticoagulants, antiplatelet agents, sedatives, an ansiolitic agent, a peptidic agent, a biopolymer agent, an antineoplastic agent, a laxative, an antidiarrheal agent, an antimicrobial agent, an antifungal agent, a vaccine, a protein, or a nucleic acid. In further aspects, the pharmaceutically active agents are selected from the group consisting of coumarins, albumins, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; xanthines such as theophylline and doxophylline; beta-2-agonist bronchodilators such as salbutamol, penterol, clenbuterol, bambuterol, salmeterol, fenoterol; anti-inflammatory agents including antiasthmatic anti-inflammatory agents, anti-arthritic anti-inflammatory agents, and non-steroidal anti-inflammatory agents, for example, sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxen, acetaminophen, ibuprofen, ketoprofen, and piroxicam; Analgesics, such as salicylates; calcium channel blockers, such as nifedipine, amlodipine, and nicardipine; angiotensin-converting enzyme inhibitors, such as captopril, benazepril hydrochloride, fosinopril sodium, trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride, and moexipril hydrochloride; beta-blockers (i.e., beta adrenergic blockers), such as sotalol hydrochloride, timolol maleate, esmolol hydrochloride, carteolol, propanolol hydrochloride, betaxolol hydrochloride, penbutolol sulfate, metoprolol tartrate, metoprolol succinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprolol fumarate; Centrally active alpha-2-agonists, such as clonidine; alpha-1-antagonists, such as doxazosin and prazosin; anticholinergics/spasmodics, such as dicyclomine hydrochloride, scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate, and oxybutynin; vasopressin analogues, such as vasopressin and desmopressin; antiarrhythmics, such as quinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamil hydrochloride, propafenone hydrochloride, flecainide acetate, procainamide hydrochloride, moricizine hydrochloride, and disopyramide phosphate; Antiparkinsonian agents, such as dopamine, L-dopa/carbidopa, selegiline, dihydroergocriptine, pergolide, lisuride, apomorphine, and bromocriptine; antianginal agents and antihypertensives, such as isosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol, and verapamil; anticoagulants and antiplatelet agents, such as coumadin, warfarin, acetylsalicylic acid, and ticlopidine; sedatives, such as benzodiazapines and barbiturates; ansiolytic agents, such as lorazepam, bromazepam, and diazepam; Peptidic and biopolymeric agents, such as calcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin, insulin, somatostatin, protirelin, interferon, desmopressin, somatotropin, timopentin, pidotimod, erythropoietin, interleukins, melatonin, granulocyte/macrophage-CSF, and heparin; antineoplastic agents, such as etoposide, etoposide phosphate, cyclophosphamide, methotrexate, 5-fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase, altretamine, mitotane, and procarbazine hydrochloride; Laxatives, such as senna concentrate, casaantranol, bisacodyl, and sodium picosulfate; antidiarrheal agents, such as diphenoxine hydrochloride, loperamide hydrochloride, furazolidone, diphenoxylate hydrochloride, and antimicrobials; vaccines, such as bacterial and viral vaccines; antibacterials, such as penicillins, cephalosporins, and macrolides, antifungal agents, such as imidazole and triazolic derivatives; and nucleic acids, such as biological proteins, and DNA sequences encoded by antisense oligonucleotides.

Anticancer agents include alkylating agents, platinum agents, antimetabolites, topoisomerase inhibitors, antineoplastic antibiotics, antimitotic agents, aromatase inhibitors, thymidylate synthase inhibitors, DNA antagonists, farnesyltransferase inhibitors, pump inhibitors, histone acetyltransferase inhibitors, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNF alpha agonists/antagonists, endothelin A receptor antagonists, retinoic acid receptor agonists, immunomodulators, hormonal and antihormonal agonists, photodynamic agents, and tyrosine kinase inhibitors.

Antibiotics include aminoglycosides (e.g., gentamicin, tobramycin, netilmicin, streptomycin, amikacin, neomycin), bacitracin, corbapenems (e.g., imipenem/cislastatin, cephalosporins, colistin, methenamine, monobactams (e.g., aztreonam), penicillins (e.g., penicillin G, penicillin V, methicillin, natcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, and vancomycin; and bacteriostatic agents such as chloramphenicol, clindanyan, macrolides (e.g., erythromycin, azithromycin, clarithromycin), lincomyan, nitrofurantoin, sulfonamides, tetracyclines (e.g., tetracycline, doxycycline, minocycline, demeclocyline, and trimethoprim. Also included are metronidazole, fluoroquinolones, and ritampin.

Enzyme inhibitors are substances that inhibit enzymatic reactions. Examples of enzyme inhibitors include edrophonium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine, tacrine, 1-hydroxymaleate, iodotubercidine, p-bromotetramisole, 10-(alpha-diethylaminopropionyl)-phenothiazine hydrochloride, calmidazolium chloride, hemicholinium-3,3,5-dinitrocatechol, diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II, 3-phenylpropagylamine, N°-monomethyl-L-arginine acetate, carbidopa, 3-hydroxybenzylhydrazine, hydralazine, chlorgyline, deprenyl, hydroxylamine, iproniazid phosphate, 6-MeO-tetrahydro-9H-pyrido-indole, nialamide, pargyline, quinacrine, semicarbazide, tranylcypromine, N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, 3-isobutyl-1-methylxanthine, papaverine, indomethacind, 2-cyclooctyl-2-hydroxyethylamine hydrochloride, 2,3-dichloro-a-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride, p-aminoglutethimide, p-aminoglutethimide tartrate, 3-iodotyrosine, alpha-methyltyrosine, acetazolamide, dichlorphenamide, Contains 6-hydroxy-2-benzothiazolesulfonamide, and allopurinol.

Antihistamines include pyrilamine, chlorpheniramine, and tetrahydrazoline, among others.

Anti-inflammatory agents include corticosteroids, nonsteroidal anti-inflammatory drugs (e.g., aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, and fenamate), acetaminophen, phenacetin, gold salts, chloroquine, D-penicillamine, methotrexate colchicine, allopurinol, probenecid, and sulfinpyrazone.

Muscle relaxants include mephenesine, methocarbomal, cyclobenzaprine hydrochloride, trihexylphenidyl hydrochloride, levodopa/carbidopa, and biperiden.

Antispasmodics include atropine, scopolamine, oxyphenonium, and papaverine.

Analgesics include aspirin, phenylbutazone, idomethacin, sulindac, tolmetic, ibuprofen, piroxicam, fenamate, acetaminophen, phenacetin, morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids (e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate, loperamide, morphine sulfate, noscapine, norcodeine, normorphine, thebaine, norbinaltorpimine, buprenorphine, clomaltrexamine, funaltrexamion, nalbuphine, nalorphine, naloxone, naloxoneazine, naltrexone, and naltrindole), procaine, lidocaine, tetracaine, and dibucaine.

Eye drops include sodium fluorescein, rose bengal, methacholine, adrenaline, cocaine, atropine, alpha-chymotrypsin, hyaluronidase, betaxalol, pilocarpine, timolol, timolol salts, and combinations thereof.

Prostaglandins are known in the art and are a class of naturally occurring, chemically related long-chain hydroxy fatty acids that have a variety of biological effects.

Antisedatives are substances that can prevent or alleviate depression. Examples of antisedatives include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazide.

Trophic factors are factors that, when present continuously, enhance the survival or lifespan of cells. Trophic factors include, but are not limited to, platelet-derived growth factor (PDGP), neutrophil-activating protein, monocyte chemoattractant protein, macrophage-inflammatory protein, platelet factor, platelet basic protein, and melanoma growth stimulating factor; epidermal growth factor, transforming growth factor (alpha), fibroblast growth factor, platelet-derived endothelial cell growth factor, insulin-like growth factor, glial-derived growth neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, bone growth/chondrogenesis-inducing factor (alpha and beta), bone morphogenetic protein, interleukins (e.g., interleukin inhibitors or interleukin receptors including interleukin 1 to interleukin 10), interferons (e.g., interferon alpha, beta, and gamma), hematopoietic factors including erythropoietin, granulocyte colony stimulating factor, macrophage colony stimulating factor, and granulocyte-macrophage colony stimulating factor; tumor necrosis factor, and transforming growth factor (beta) including beta-1, beta-2, beta-3, inhibin, and activin.

Hormones include estrogens (e.g., estradiol, estrone, estriol, diethylstibestrol, quinestrol, chlorotrianisene, ethinyl estradiol, mestranol), antiestrogens (e.g., clomiphene, tamoxifen), progestins (e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone, norgestrel), antiprogestins (mifepristone), androgens (e.g., testosterone cypionate, fluoxymesterone, danazol, testolactone), antiandrogens (e.g., cyproterone acetate, flutamide), thyroid hormones (e.g., triiodothyronne, thyroxine, propylthiouracil, methimazole, and iodixode), and pituitary hormones (e.g., Hormones include corticotropin, sumutotropin, oxytocin, and vasopressin. Hormones are commonly employed for hormone replacement therapy and/or birth control purposes. In addition, steroid hormones, such as prednisone, are used as immunosuppressants and anti-inflammatory agents.

In some embodiments, the additive is an agent that stimulates tissue formation and/or repair and regrowth of native tissue, and any combination thereof. Agents that increase the formation of new tissue at the site of injection and/or stimulate repair or regrowth of native tissue include, but are not limited to, osteogenic factors including fibroblast growth factor (FGF), transforming growth factor-beta (TGF-β, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), connective tissue-activated peptide (CTAP), bone morphogenetic protein, heparin, angiotensin II (A-II) and fragments thereof, insulin-like growth factor, tumor necrosis factor, interleukins, colony stimulating factors, erythropoietin, nerve growth factor, interferons, biologically active analogues, fragments and derivatives of such growth factors, and any combination thereof.

In some embodiments, the silk composition can further comprise at least one additional material for enhancing elastic tissue, such as a dermal filler material, including but not limited to poly(methyl methacrylate) microspheres, hydroxylapatite, poly(L-lactic acid), collagen, elastin, and glycosaminoglycans, hyaluronic acid, commercial dermal filler products such as BOTOX® (from Allergan), DYSPORT®, COSMODERM®, EVOLENCE®, RADIESSE®, RESTYLANE®, JUVEDERM® (from Allergan), SCULPTRA®, PERLANE®, and CAPTIQUE®, and any combinations thereof.

In some embodiments, the additive is a wound healing agent. As used herein, a "wound healing agent" is a compound or composition that actively promotes the wound healing process. Exemplary wound healing agents include, but are not limited to, dexpanthenol; growth factors; enzymes, hormones; povidone-iodide; fatty acids; anti-inflammatory agents; antibiotics; antimicrobial agents; antiseptics; cytokines; thrombin; analgesics; opioids; aminoxyl; furoxal; nitrosothiols; nitrates and anthocyanins; nucleosides, such as adenosine; and nucleotides, such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP); neurotransmitters/neuromodulators, such as acetylcholine and 5-hydroxytryptamine (serotonin/5-HT); histamine and catecholamines, such as adrenaline and noradrenaline; lipid molecules such as sphingosine-1-phosphate and lysophosphatidic acid; amino acids such as arginine and lysine; peptides such as bradykinin, substance P, and calcium gene-related peptide (CGRP); nitric oxide; and any combination thereof.

In certain embodiments, the active agent described herein is an immunogen. In one embodiment, the immunogen is a vaccine. Most vaccines are sensitive to environmental conditions under which they are stored and/or transported. For example, freezing can increase the loss of reactogenicity (e.g., the ability to elicit an immunological response) and/or potency for some vaccines (e.g., HepB, and DTaP/IPV/HIB), or can cause hairline cracks in the container, leading to contamination. Additionally, some vaccines (e.g., BCG, varicella, and MMR) are heat sensitive. Many vaccines (e.g., BCG, MMR, varicella, Meningococcal C conjugate, and most DTaP-containing vaccines) are light sensitive. See, e.g., Galazka et al., Thermostability of vaccines, in Global Programme for Vaccines & Immunization (World Health Organization, Geneva, 1998); See Peetermans et al., Stability of freeze-dried rubella virus vaccine (Cendehill strain) at various temperatures, 1 J. Biological Standardization 179 (1973). Thus, the compositions and methods described herein also provide for stabilization of vaccines independent of cold chain and/or other environmental conditions.

In some embodiments, the additive is a cell, e.g., a biological cell. Cells that are readily incorporated into the composition may be derived from any source, e.g., mammalian, insect, plant, etc. In some embodiments, the cells may be human, rat, or mouse cells. In general, cells to be used with the compositions described herein may be any type of cell. In general, the cells must be viable when encapsulated in the composition. In some embodiments, cells that may be used with the composition include, but are not limited to, mammalian cells (e.g., human cells, primate cells, mammalian cells, rodent cells, etc.), avian cells, fish cells, insect cells, plant cells, fungal cells, bacterial cells, and hybrid cells. In some embodiments, exemplary cells that may be used with the composition may be platelets, activated platelets, stem cells, totipotent cells, pluripotent cells, and/or embryonic stem cells. In some embodiments, exemplary cells that may be encapsulated in the composition include, but are not limited to, primary cells and/or cell lines from any tissue. For example, cardiomyocytes, myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells (e.g., monocytes, neutrophils, macrophages, etc.), ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, sperm cells, oocytes, liver cells, lung epithelial cells, digestive tract epithelial cells, intestinal epithelial cells, liver, skin epithelial cells, and/or combinations thereof can be included in the silk/platelet compositions disclosed herein. Those of ordinary skill in the art will recognize that the cells listed herein are exemplary and do not represent an exhaustive list of cells. The cells can be obtained from a donor (allogeneic) or a recipient (autologous). The cells can be obtained by, but are not limited to, biopsy means or other surgical means known to those of ordinary skill in the art.

In some embodiments, the cell is a genetically modified cell. The cell is genetically modified to be able to secrete and excrete a compound of interest, such as a bioactive agent, a growth factor, a differentiation factor, a cytokine, etc. Methods for causing a genetically modified cell to secrete and excrete a compound of interest are known in the art and can be readily adapted by one of ordinary skill in the art.

Additionally, differentiated cells reprogrammed into stem cells can be used. For example, human skin cells have been reprogrammed into embryonic stem cells by transduction of Oct3/4, Sox2, c-Myc and Klf4 (reviewed in Junying Yu, et. al., Science, 2007, 318, 1917-1920 and Takahashi K. et. al., Cell, 2007, 131, 1-12).

In some embodiments, the additive may be silk fibroin particles. Silk fibroin particles and methods for making them are described herein above.

In some embodiments, the additive can be a silk-based material. The silk-based material can be selected from the group consisting of silk fibers, fine-sized silk fibers, unprocessed silk fibers, silk particles, and any combination thereof. In some embodiments, the additive is a silk fiber. The use of silk fibers is disclosed, for example, in U.S. Patent Application Publication No. US20110046686, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the silk fibers are microfibers or nanofibers. In some embodiments, the additive is a micron-sized (10-600 μm) silk fiber. Micron-sized silk fibers can be obtained by hydrolyzing refined silk fibroin or by increasing the boiling time of the refinement process. Alkaline hydrolysis of silk fibroin to obtain micron-sized silk fibers is disclosed, for example, in Mandal et al., PNAS, 2012, doi: 10.1073/pnas.1119474109; U.S. Provisional Application No. 61/621,209, filed April 6, 2012; and PCT Application No. PCT/US13/35389, filed April 5, 2013, the entire contents of which are incorporated herein by reference. Since regenerated silk fibers prepared from HFIP silk solutions are mechanically strong, regenerated silk fibers can be used as an additive.

In some embodiments, the silk fiber is a raw silk fiber, e.g., raw silk or raw silk fiber. The term "raw silk" or "raw silk fiber" refers to a silk fiber that has not been treated to remove sericin, and thus encompasses, for example, silk fibers harvested directly from cocoons. Thus, raw silk fiber refers to silk fibroin obtained directly from the silk gland. When silk fibroin obtained directly from the silk gland is allowed to dry, its structure is referred to as silk I in the solid state. Thus, raw silk fiber comprises silk fibroin that is predominantly in the silk I conformation. On the other hand, regenerated or processed silk fiber comprises silk fibroin having a significant degree of silk II or beta-sheet crystallinity.

In some embodiments, the additive is a biocompatible polymer. Exemplary biocompatible polymers include, but are not limited to, polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polyesters, poly(ortho esters), poly(phosphazines), poly(phosphate esters), polycaprolactone, gelatin, collagen, fibronectin, keratin, polyaspartic acid, alginates, chitosan, chitin, hyaluronic acid, pectin, polyhydroxyalkanoates, dextrans, and polyanhydrides, polyethylene oxide (PEO), poly(ethylene glycol) (PEG), triblock copolymers, polylysine, alginates, polyaspartic acid, any derivatives thereof, and any combinations thereof. Other exemplary biocompatible polymers that may be used in accordance with the present disclosure include, but are not limited to, those disclosed in, for example, US Pat. Nos. 6,302,848; 6,395,734; Nos. 6,127,143; 5,263,992; 6,379,690; 5,015,476; 4,806,355; 6,372,244; 6,310,188; 5,093,489; US 387,413; 6,325,810; 6,337,198; US 6,267,776; 5,576,881; 6,245,537; 5,902,800; and 5,270,419, the entire contents of which are incorporated herein by reference in their entireties. The term "biocompatible" as used herein refers to a material that does not induce a substantial immune response in a host.

In some embodiments, the biocompatible polymer is PEG or PEO. As used herein, the term "polyethylene glycol" or "PEG" is an ethylene glycol polymer containing from about 20 to about 2,000,000 linked monomers, typically from about 50 to 1,000 linked monomers, and usually from about 100 to 300. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. In general, PEG, PEO, and POE refer to the same chemically, but historically, PEG has tended to refer to polymers and oligomers having a molecular mass less than 20,000 g/mol, PEO has tended to refer to polymers having a molecular mass greater than 20,000 g/mol, and POE has tended to refer to polymers having any molecular mass. PEG and PEO are liquids or low melting point solids, depending on their molecular weight. PEG is prepared by the polymerization of ethylene oxide and is commercially available over a wide range of molecular weights, from 300 g/mol to 10,000,000 g/mol. PEGs and PEOs of different molecular weights find use in different applications and have different physical properties (e.g. viscosity) due to chain length effects, but their chemical properties are nearly identical. Different forms of PEG are also available, depending on the initiator used for the polymerization process - the most common initiators being monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), the shortened form mPEG. Lower molecular weight PEGs are also available as purer oligomers, referred to as monodisperse, or homogeneous or discrete PEGs are also available in different geometries.

As used herein, the term PEG is intended to be inclusive rather than exclusive. The term PEG includes poly(ethylene glycol) in any form, including alkoxy PEG, difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendant PEG (i.e., a related polymer or PEG having one or more functional groups attached to the polymer backbone), or PEG having a degradable linkage. Additionally, the PEG backbone can be linear or branched. Branched polymer backbones are generally well known in the art. Typically, a branched polymer has a central branched core moiety and a plurality of linear polymer chains linked to the polymerized branched core. PEG is commonly used in a branched form, which can be prepared by adding ethylene oxide to various polyols, such as glycerol, pentaerythritol, and sorbitol. The central branched moiety can also be derived from a number of amino acids, such as lysine. Branched poly(ethylene glycol)s can be represented by the general form R(-PEG-OH)m, where R represents a core moiety such as glycerol or pentaerythritol, and m represents the number of arms. Multiarm PEG molecules, such as those described in U.S. Pat. No. 5,932,462, the entire contents of which are incorporated herein by reference, can also be used as biocompatible polymers.

Some exemplary PEGs include, but are not limited to, PEG20, PEG30, PEG40, PEG60, PEG80, PEG100, PEG115, PEG200, PEG 300, PEG400, PEG500, PEG600, PEG1000, PEG1500, PEG2000, PEG3350, PEG4000, PEG4600, PEG5000, PEG6000, PEG8000, PEG11000, PEG12000, PEG15000, PEG 20000, PEG250000, PEG500000, PEG100000, PEG2000000, and the like. In some embodiments, the PEG has a MW of 10,000 daltons. In some embodiments, the PEG has a MW of 100,000, i.e., is PEO with a MW of 100,000.

In some embodiments, the additive is an enzyme that hydrolyzes silk fibroin. Without wishing to be bound by theory, such enzymes can be used to control the degradation of manufactured articles.

In some embodiments, the additive is a plasticizer. Without wishing to be bound by theory, the inclusion of a plasticizer may affect the flexibility and/or solubility of the silk-based article, e.g., a silk fibroin film. In one embodiment, the plasticizer is glycerol. Silk-based materials, e.g., silk films comprising glycerol, are described in WO 2010/042798, which is incorporated herein by reference in its entirety.

Examples of other additives include, but are not limited to, cell adhesion mediators known to affect cell adhesion (Schaffner P & Dard, Cell Mol Life Sci., 2003, 60(1): 119-32 and Hersel U. et al., Biomaterials, 2003, 24(24):4385-415), such as collagen, elastin, fibronectin, vitronectin, laminin, proteoglycans, or peptides containing known integrin binding domains, e.g., the "RGD" integrin binding sequence, or modifications thereof; biologically active ligands; and agents that enhance or inhibit specific variants of cell or tissue ingrowth.

In some embodiments, the release of the active agent from the silk fibroin composition can be controlled by adding an additive to the composition that can interfere with interactions between the silk fibroin and the active agent. Thus, in some embodiments, the additive is an agent that interferes, inhibits, or reduces protein-active agent interactions. For example, silk fibroin has a net negative charge. Thus, an active agent having a net positive charge can interact with the silk fibroin through ionic/electrostatic interactions and be retained in the composition. The rate of release of the agent from the composition can be altered by altering the ionic/electrostatic interactions. One way to alter the ionic/electrostatic interactions can be by adding a cationic molecule to the composition. Some other agents can interact with the silk fibroin through hydrophobic interactions. Without wishing to be bound by theory, it is believed that such agents can be released using an additive that interferes, inhibits, or reduces non-ionic interactions. Thus, in some embodiments, the additive can be a surfactant. As used herein, the term "surfactant" refers to a natural or synthetic amphiphilic compound. Surfactants can be non-ionic, zwitterionic, or ionic. Non-limiting examples of surfactants include polysorbates such as polysorbate 20 (TWEEN® 20), polysorbate 40 (TWEEN® 40), polysorbate 60 (TWEEN® 60), polysorbate 61 (TWEEN® 61), polysorbate 65 (TWEEN® 65), polysorbate 80 (TWEEN® 80), and polysorbate 81 (TWEEN® 81); Poloxamers (polyethylene-polypropylene copolymers), such as poloxamer 124 (Pluronic® L44), poloxamer 181 (Pluronic® L61), poloxamer 182 (Pluronic® L62), poloxamer 184 (Pluronic® L64), poloxamer 188 (Pluronic® F68), poloxamer 237 (Pluronic® F87), poloxamer 338 (Pluronic® L108), poloxamer 407 (Pluronic® F127), polyoxyethylene glycol dodecyl ethers, such as BRIZE® 30, and BRIZE® 35; 2-dodecoxyethanol (LUBROL®-PX); polyoxyethylene octyl phenyl ether (TRITON® X-100); sodium dodecyl sulfate (SDS); 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS); 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO); sucrose monolaurate; and sodium cholate. Other non-limiting examples of surfactant excipients include those described in, for example, Pharmaceutical Dosage Forms and Drug Delivery Systems (Howard C. Ansel et al., eds., Lippincott Williams & Wilkins Publishers, ^7th ed. 1999); Remington: The Science and Practice of Pharmacy (Alfonso R. Gennaro ed., Lippincott, Williams & Wilkins, ^20th ed. 2000); Goodman &Gilman's The Pharmacological Basis of Therapeutics (Joel G. Hardman et al., eds., McGraw-Hill Professional, ^10th ed. 2001); and Handbook of Pharmaceutical Excipients (Raymond C. Rowe et al., APhA Publications, 4th edition 2003), each of which is incorporated herein by reference in its entirety. In some embodiments, the surfactant is a cationic polymer. In one embodiment, the cationic polymer is a polylysine, e.g., ε-poly-L-lysine. In an alternative embodiment, the surfactant is an anionic polymer. In one embodiment, the anionic polymer is a polyglutamate, e.g., poly-L-glutamate.

For administration to a subject, the silk fibroin compositions provided can be formulated in a pharmaceutically acceptable composition comprising the silk fibroin composition disclosed herein, and can be formulated with one or more pharmaceutically acceptable carriers (additives) and/or diluents. For administration to a subject, the pharmaceutical compositions can generally be formulated in unit dosage form. Additionally, the pharmaceutical compositions can be in solid or liquid form for administration, such as (1) topical application, for example, as a cream, ointment, controlled-release patch, or spray applied to the skin; (2) parenteral administration, for example, as a sterile solution or suspension, or as a sustained-release formulation, by subcutaneous, intramuscular, intravenous, or epidural injection; (3) oral administration, for example, as a drench (aqueous or non-aqueous solution or suspension) for application to the tongue, rosin, dragees, capsules, pills, tablets (e.g., intended for buccal, sublingual, and total absorption), boluses, powders, granules, pastes; (4) vaginally or rectally, for example, as a pessary, cream, or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8) transmucosally; or (9) nasally. The compositions may also be implanted into the patient or injected using a drug delivery composition. See, e.g., Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981); U.S. Patent No. 3,773,919; and U.S. Patent No. 35 3,270,960.

As used herein, the term "pharmaceutically acceptable" means a compound, material, composition, and/or dosage form that is within the scope of sound medical judgment and suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problem or complication, and commensurate with a reasonable benefit/risk ratio.

As used herein, the term "pharmaceutically-acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., a lubricant, talc magnesium, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, which is included to carry or deliver the title compound from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense that it can be combined with the other ingredients of the formulation without being deleterious to the patient. Some examples of materials that serve as pharmaceutically-acceptable carriers include (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweetening agents, flavoring agents, perfumes, preservatives and antioxidants may also be present in the formulation. The terms "excipient", "carrier", "pharmaceutically acceptable carrier" and the like are used interchangeably herein.

Pharmaceutically acceptable antioxidants include, but are not limited to, (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lectisin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

As used herein, the term "administered" means placement of a drug delivery composition into a subject by a route or method that results in at least partial localization of the pharmaceutically active agent at the desired site.

The composition may be administered by any suitable route that results in effective treatment in the subject, i.e., administration results in delivery of at least a portion of the pharmaceutically active agent to a desired location in the subject to which it is being delivered. Exemplary modes of administration include, but are not limited to, topical, implant, injection, infusion, instillation, implantation, or ingestion. "Injection" includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracerebroventricular, intrathecal, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebral spinal, and intrasternal injection and infusion.

Injectable formulations require that the composition meet certain physical criteria for administration by injection. For example, such formulations require a sufficient level of so-called needle penetration (syringeability). The term needle penetration refers to the time and force required for manual injection (or the time required for injection using an autoinjector), which are important parameters that must be respected as they may affect the usability of the product to the end user. The force required to inject a solution at a given injection rate through a needle of a predetermined gauge and length is referred to as needle penetration (Burckbuchler, V et al., Eur. J.Pharm. Biopharm. 2010, 76 (3): 351-356). The Hagen-Poisel equation can be used to estimate the travel (or glide) force, which can be expressed as:

F = 8QμL/πR4 x A (Equation 1)

(where, Q = volumetric flow rate; μ = fluid viscosity; L = needle length; R = needle inside diameter; A = cross-sectional area of the syringe plunger; F = frictionless moving force).

The viscosity of a solution depends on the protein itself, protein concentration, temperature and formulation, e.g. pH, type of excipients and concentration of excipients. In addition to the protein itself, protein concentration is another important factor for viscosity. Subcutaneous injections are generally limited to injection volumes of approximately < 1.5 mL (see, e.g., Adler (2012) Am. Pharmaceutical Rev. pp. 1-9).

In some embodiments, the silk fibroin compositions disclosed herein can be implanted into a subject. As used herein, the term "implanted" and grammatically related terms refer to temporary, semi-permanent, or permanent positioning of a composition at a particular location within a subject. The term does not require permanent fixation of the composition to a particular location or site. Exemplary in vivo locations include, but are not limited to, sites of wounds, trauma, or disease.

For administration to a subject, the silk fibroin compositions can generally be formulated in a unit dosage injectable form. Compositions and preparations suitable for injection include sterile aqueous solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, cell culture medium, a buffer (e.g., phosphate buffered saline), a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof. In some embodiments, the pharmaceutical carrier can be a buffered solution (e.g., PBS).

In some embodiments, the pharmaceutical composition can be administered by a delivery device, e.g., a syringe. Thus, a further aspect described herein provides a delivery device comprising at least one chamber having an outlet, wherein the at least one chamber contains a predetermined amount of any of the compositions described herein, and the outlet provides an outlet for the composition isolated within the chamber. In some embodiments, the delivery device described herein further comprises an actuator to control release of the composition through the outlet. Such a delivery device can be any device that facilitates administration of any of the compositions described herein to a subject, e.g., a syringe, a dry powder syringe, a nasal spray, a nebulizer, or an implant such as a microchip, for sustained-release or controlled-release of any of the compositions described herein.

Kit

Also provided herein are sample collection devices and kits that can be employed in the compositions and methods described herein. Accordingly, another aspect provided herein is a sample collection device comprising a chamber for collecting a sample, for example a biological sample or an active agent, wherein the chamber comprises a silk fibroin composition. Upon contact of the biological sample or the active agent with the silk fibroin solution, at least one property of the biological sample or at least one bioactivity of the active agent can be stabilized for a period of time. In some embodiments, the silk fibroin composition present in the chamber can be in the form of a solution, a powder, a suspension, or a gel.

Any sample known in the art, for example, a biological sample collection device, may be applied herein. Non-limiting examples of devices may include a biological specimen collection tube (e.g., a blood collection tube), a vial, a syringe, a centrifuge tube, a microtiter plate, a dipstick, a biological specimen collection bag, a sponge, and any combination thereof. In one embodiment, the device may be a blood collection tube or bag.

In some embodiments, the chamber of the device can further comprise an additive as described herein. In some embodiments, the chamber of the device can further comprise an anti-coagulant, a stabilizer, a gelling-inducing agent, a protease or digestive enzyme, or any combination thereof. Stabilizers that can be used to further enhance stabilization of the target component of the biological sample can include, but are not limited to, saccharides, sugar alcohols, ions, surfactants, amino acids, human serum albumin, bovine serum albumin, gelatin, and gelatin derivatives, antioxidants, any of the stabilizers described herein, or any combination thereof. In some embodiments, the stabilizer can be a nuclease inhibitor or a proteinase inhibitor. In some embodiments, RNA is detected from the biological sample, and the stabilizer can comprise an RNase inhibitor and/or DEPC.

The gelation-inducing agent can be any agent capable of inducing gelation of silk fibroin to form a silk-based material, such as a functionally-activated PEG as described in International Application No. WO/2012/031144, the contents of which are incorporated herein by reference. For example, in one embodiment, the chamber can further comprise a first functionally-activated PEG component as described in International Application No. WO/2012/031144. In such an embodiment, the biological sample collection device further comprises an additional chamber comprising a second functionally-activated PEG component that can react with the first functionally-activated PEG component to form a solid-state silk-based material.

In some embodiments where the device is a blood collection tube, the chamber can further comprise at least one additive commonly found in conventional blood collection tubes, including but not limited to, a clot activator, sodium heparin, lithium heparin, a thrombin-based clot activator, K ₂ EDTA, liquid K ₃ EDTA, Na ₂ EDTA, potassium oxalate, sodium fluoride, sodium polyanethol sulfonate (SPS), an acid citrate dextrose additive (ACD) (including trisodium citrate and citric acid), a mixture comprising sodium citrate, citrate, theophylline, adenosine, dipyridamole (CTAD), or any combination thereof.

Also provided herein are kits comprising one or more embodiments of a biological sample collection device as described herein. In some embodiments, the kit can further comprise at least one container containing a gelling-inducing agent, a pH-reducing agent (e.g., an acid), or a combination thereof, including, for example, a functionally activated PEG moiety as disclosed in International Application No. WO/2012/031144.

In some embodiments, the kit can further comprise a container containing a silk-solubilizing agent. Examples of silk-solubilizing agents can include water, a buffer solution, or a combination thereof. In some embodiments, the target component to be detected is not a protein or a peptide, and the silk-solubilizing agent can include a proteinase that degrades silk fibroin.

In some embodiments, the kit can further comprise a container containing a stabilizing agent, wherein the stabilizing agent stabilizes at least one component of the biological sample. For example, the stabilizing agent can include, but is not limited to, a saccharide, a sugar alcohol, an ion, a surfactant, an amino acid, human serum albumin, bovine serum albumin, gelatin, and gelatin derivatives, an antioxidant, any of the stabilizing agents described herein, or any combination thereof. In one embodiment, the stabilizing agent can be a nuclease inhibitor or a proteinase inhibitor. In one embodiment, RNA is detected from the biological sample, and the stabilizing agent can include an RNase inhibitor.

In some embodiments, the kit can further comprise a container containing an agent for detecting at least one component of the biological sample. For example, depending on the purpose of the kit for genotyping or expression analysis, one skilled in the art can determine an appropriate agent for performing the analysis. By way of example only, the agent can include a component-purifying agent (e.g., an agent that purifies a target component, such as RNA, from a non-target component, such as genomic DNA; or an extract protein from a biological sample); a nucleic acid amplification agent (e.g., but not limited to, primers, polymerases, oligonucleotides); an immuno-affinity-based detection agent (e.g., but not limited to, primary and/or secondary antibodies, and aptamers), a labeling agent (e.g., a fluorescent dye, an agent for a colorimetric enzyme-substrate reaction, such as horseradish peroxidase (HRP), and a separate chromogenic substrate (e.g., TMB, DAB, ABTS), or any combination thereof.

In some embodiments of the compositions described herein, the silk fibroin can be modified to control its degradation and thus the release of the active agent, for example, where the release occurs over a period of time ranging from hours to days, or months. In some embodiments, the compositions described herein can be combined with other types of delivery systems known and available to those skilled in the art. These include, for example, polymer-based systems such as polylactic acid and/or polyglycolic acid, polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and/or combinations thereof. Microcapsules of the aforementioned polymer-containing drugs are described, for example, in U.S. Pat. No. 5,075,109. Other examples include non-polymeric systems that are lipid-based, including sterols such as cholesterol, cholesterol esters, and fatty acids or neuca 1 fats such as mono-, di-, and triglycerides; hydrogel release systems; liposome-based systems; phospholipid-based systems; Silastic systems; peptide-based systems; or partially fused implants. Specific examples include, but are not limited to, erosion systems in which the composition is contained in one form within a matrix (e.g., as described in U.S. Pat. Nos. 4,452,775, 4,675,189, 5,736,152, 4,667,014, 4,748,034, and -29 5,239,660), or diffusion systems in which the active ingredient is released at a controlled rate (e.g., as described in U.S. Pat. Nos. 3,832,253, 3,854, 480, 5,133,974, and 5,407,686). The formulations may be, for example, microspheres, hydrogels, polymer reservoirs, cholesterol matrices, or polymer systems. In some embodiments, the system may also allow sustained or controlled release of the composition to occur, for example, by controlling the diffusion or erosion/degradation rate of a formulation containing the composition. Additionally, a pump-based hardware delivery system may be used to deliver one or more embodiments of a composition or formulation described herein. The use of extended-release formulations or implants is particularly suited for the treatment of chronic conditions, such as diabetes. Extended-release, as used herein, means that the formulation or implant is constructed and arranged to deliver a composition or formulation described herein at therapeutic levels for at least 30 days, or at least 60 days. In some embodiments, extended-release means that the formulation or implant is configured to deliver therapeutic levels of the active agent for several months.

Specific exemplary applications

According to various aspects of the embodiments described herein, the formation of a silk-based material comprising silk fibroin and a biological sample can stabilize at least one component of the biological sample, which can later enable detection and/or analysis of the component. In some embodiments, such silk-based materials comprising a biological sample can be stored and transported without refrigeration or freezing, which are typical methods for storing and processing biological samples to maintain/preserve sample quality for diagnostic assessments of human health. Accordingly, in some embodiments, the silk-based materials and methods described herein can be useful for diagnostic applications. For example, the silk-based materials and methods can be used to maintain and/or preserve the quality of a biological sample during storage and/or transport or in some developing countries or remote field conditions that do not have the minimum infrastructure to support continuous refrigerated storage, so that at least one component of the biological sample can be analyzed for diagnostic applications. In some embodiments, the silk-based materials and methods described herein can be used to maintain and/or retain the quality of biological samples under at least one or any combination of the following conditions previously described: (a) a temperature above 0° C. (e.g., at least about room temperature or higher) during storage and/or transport; (b) exposure to light (e.g., UV, infrared, and/or visible light) during storage and/or transport; and (c) a relative humidity of at least about 10% or higher during storage and/or transport.

Accordingly, another aspect provided herein relates to a method for using a silk-based material as described herein. The method comprises: (a) providing one or more embodiments of a silk-based material comprising silk fibroin and a biological sample; and (b) analyzing at least one component of the biological sample at least once.

In some embodiments, the silk-based material can be formed by contacting a biological sample with silk fibroin. Methods of forming a silk-based material are described herein. In some embodiments, the silk-based material can be in a liquid state. In other embodiments, the silk-based material can be a degradable solid-state material (such as but not limited to a soluble silk-based film, or foam). In some embodiments, a subject or patient in need of diagnosis of a disease or disorder can provide a biological sample, and the biological sample can be contacted with silk fibroin to form a silk-based material, which is then sent to a diagnostic testing laboratory for analysis. In some embodiments, the biological sample can be collected from the subject by a skilled practitioner in a clinical setting, and the biological sample can then be contacted with silk fibroin to form a silk-based material, which is then sent to a diagnostic testing laboratory for analysis.

In some embodiments, at least one component of a biological sample can be detected and/or analyzed without isolating the component from the biological sample, and in alternative embodiments, the component can be extracted or recovered from at least a portion of the silk-based material prior to detection and/or analysis using any method known in the art. According to some embodiments of the silk-based material described herein, the silk-based material can be soluble in an aqueous solution (e.g., water, a buffer solution, or a combination thereof). Unlike cellulose-based technologies, such as dried blood spots where blood is absorbed into filter paper and difficult to recover, at least a portion of the degradable silk-based material described herein can be soluble in an aqueous solution (e.g., water, a buffer solution, or a combination thereof). The resulting silk/biological sample solution can then be subjected to liquid assays without further purification, such as routine ELISA and Luminex™ assays described in the Examples. Thus, in some embodiments, the method can further comprise contacting at least a portion of the silk-based material with an aqueous solution (e.g., water, a buffer solution, or a combination thereof) prior to analyzing at least one component of the biological sample.

Various types of analyses can be performed on the active agent/target component of the biological sample, for example, depending on the nature of the agent/component. Non-limiting examples of analyses include, but are not limited to, genotyping, nucleic acid sequencing, expression analysis (e.g., protein level, or transcript level), binding affinity, enzymatic activity, transfection efficiency, cell counting, cell identification, cell viability, immunogenicity, infectivity, metabolite profiling, and any combination thereof. In some embodiments, at least one component of the biological sample is analyzed according to at least one of genotyping or nucleic acid sequencing, expression analysis (e.g., protein level and/or transcript level), metabolite profiling, or any combination thereof. Various methods for performing such analyses may include, but are not limited to, polymerase chain reaction (PCR), real-time quantitative PCR, microarray, western blot, immunohistochemical analysis, enzyme-linked absorbance assay (ELISA), mass spectrometry, nucleic acid sequencing, flow cytometry, gas chromatography, high performance liquid chromatography, nuclear magnetic resonance (NMR) spectroscopy, or any combination thereof. Techniques for nucleic acid sequencing are known in the art and can be used to assay components to determine nucleic acids or gene expression measurements, including but not limited to, for example, DNA sequencing, RNA sequencing, de novo sequencing, next generation sequencing such as massively parallel signature sequencing (MPSS), polony sequencing, pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, ion semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, sequencing by hybridization, sequencing using mass spectrometry, microfluidic Sanger sequencing, microscopy-based sequencing techniques, RNA polymerase (RNAP) sequencing, or any combination thereof.

In some embodiments, at least one analysis can be performed in a format that can interface with a reading device or system. In some embodiments, at least one analysis can be performed on the system.

In some embodiments, prior to contacting the silk-based material with the aqueous solution, the silk-based material can be reduced or aliquoted into smaller portions, some of which can be stored for later analysis and/or analyzed for different target components (e.g., proteins, nucleic acids, and/or metabolites). The silk-based material can be aliquoted into smaller portions by weight or volume.

Example

The following examples illustrate some embodiments and aspects of the present invention. It will be apparent to those skilled in the art that various modifications, additions, substitutions, etc. may be made without altering the spirit or scope of the present invention, and that such modifications and variations are included within the scope of the present invention as defined in the claims that follow. The following examples do not limit the present invention in any way.

Example 1: Exemplary materials and methods used to prepare a composition comprising silk fibroin, and their characterization

Silk fibroin solution. Silkworm cocoons were purified using a modified extraction process as described in the literature [Sofia S et al. (2001) Journal of Biomedical Materials Research, 54, 139-148]. An exemplary protocol for preparing a composition of low molecular weight silk fibroin is provided herein.

- Cut the cocoon and remove the pupa, pupa shell, and all dirt from inside the cocoon.

- Refining the pieces of kochi in a boiling sodium carbonate ( _Na2CO3 ) solution of ~0.02 _M using a refining time of approximately 60 minutes or more.

- Rinse the purified silk fibroin in water (e.g. Milli-Q water) at least three times, for at least 30 minutes each time.

- Air dry the rinsed silk fibroin.

- Dissolve silk fibroin in 9.3 M lithium bromide solution (ReagentPlus >99%, Sigma Aldrich, MO, USA) at 60°C and dialyze against water (e.g., Milli-Q water) with regular water exchange, for example, every 6 hours, for about 2 days using, for example, a Slide-A-Lyzer dialysis cassette (MWCO 3,500, Thermo Scientific, IL, USA).

- Centrifuge the prepared aqueous silk solution twice for 20 minutes each time at approximately 11,000 rpm.

- The prepared aqueous low molecular weight silk fibroin solution has a silk fibroin concentration of 7% wt/vol to 9% wt/vol. Up to this point, the silk fibroin solution is called a purified as-is solution. An additional autoclaving step can be performed on the purified as-is silk fibroin solution, after which the silk fibroin solution is called an autoclaved solution.

- Store the silk fibroin solution at 4℃.

Preparation of solid-state silk fibroin. A purified, as-is or autoclaved, silk fibroin solution is subjected to a freezing step, and a primary drying, or a combination of the primary and secondary drying steps, as schematically illustrated in FIG. 1. During the freezing step, the silk fibroin solution is frozen from room temperature to -40°C at atmospheric pressure at a rate of 0.8°C/min and maintained at -40°C for 480 minutes. During the primary drying, the silk fibroin solution is dried from -40°C to -20°C at a ramp rate of 0.2°C/min at 100 mtorr and maintained at -20°C for 2400 minutes. During the secondary drying, the silk fibroin solution is dried from -20°C to 4°C at a ramp rate of 0.2°C/min at 100 mtorr and maintained at 4°C for 620 minutes. The silk fibroin foam formed at the end of the freezing step and the first drying step is called a first drying only foam (P). The silk fibroin foam formed at the end of the freezing step, the first drying step, and the second drying step is called a first + second drying foam (P+S). The silk foam is stored at 4°C, 22°C, or 37°C. Then, according to the flow chart of Fig. 1, the silk fibroin solution can be prepared by dissolving the silk foam.

Characterization. The molecular weight distribution of silk fibroin solutions prepared from silk foams under different temperature storage conditions and boiling times was determined using gel electrophoresis. Figure 2 illustrates the method used to quantify the molecular weight distribution. The electrophoretic mobility of fibroin molecules was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For each condition, 0.15% (1.5 mg/mL) silk fibroin solution was loaded into a 3-8% Tris acetate gel (NuPAGE, Life Technologies, Grand Island, NY). The gel was run with a high molecular weight ladder as a reference (Mark 12 protein standard, Life Technologies) and stained with a colloidal blue stain kit (Life Technologies). The molecular weight distribution of the silk solutions was determined by imaging the gels, performing pixel shader analysis, and normalizing across all lanes to a single peak intensity value (ImageJ, NIH, Bethesda, MD, USA). A rectangular bracket containing a molecular weight ladder was applied to each lane and used as a reference frame. The bracket was positioned directly above the beginning of the trace and extended beyond the boundary of the lowest protein trace, the 21.5 kDa marker point.

ImageJ software automatically averages pixel intensities horizontally across the entire rectangle, then plots this average down the length of the trace (top of the trace = left, bottom of the trace = right). Using the ladder positions, it generates brackets like this:

1) Over 200 kDa (from the top of the country to the 200 kDa ladder position)

2) 200 kDa - 116 kDa

3) 116 kDa - 66.3 kDa

4) 66.3 kDa - 36.5 kDa (lowest molecular weight that can be separated)

To define the molecular weight distribution percentage for each molecular weight bracket, the area under the curve (above the minimum background boundary) within each bracket is normalized to the total area between the top and bottom of its boundary. The bracketed ranges are then plotted across samples prepared from different boiling times to semi-quantify the change in molecular weight. For example, in Figure 2b, the percentage of silk fibroin having a molecular weight greater than 200 kDa can be calculated using the following formula:

Figures 3, 4, and 5 illustrate the molecular weight distributions of silk fibroin solutions obtained from silk foams stored at 4°C, 22°C, and 37°C, respectively. Two types of silk foams were used for each storage temperature: silk foams through primary drying, and silk foams through both primary and secondary drying. For all silk foam samples tested, the percentage of silk fibroin having a molecular weight greater than 200 kDa decreased with increasing scouring time. On the other hand, the percentage of silk fibroin having a molecular weight less than 120 kDa increased with increasing scouring time. As a result of the long refining time (greater than 60 minutes), the low molecular weight silk fibroin described herein comprises a population of silk fibroin fragments having a molecular weight distribution characterized in that not more than 15% of the total number (or total moles) or total weight of the silk fibroin fragments in the population have a molecular weight greater than 200 kDa and more than 50% of the total number (or total moles) or total weight of the silk fibroin fragments in the population have a molecular weight within a specified range, wherein the specified range is from about 3.5 kDa to about 120 kDa, or from about 5 kDa to about 125 kDa.

To compare the differences in solubility of the pulverized silk powder from the freeze-dried systems depicted in Figure 1, studies were conducted by varying i) boiling time and ii) mass of powder loading according to the solubilization ratio table in Figure 1. Films were cast from each recovered solution to directly measure the silk concentration in the solution in units of wt/vol %. Following complete drying and weighing, the films were measured for secondary structure using FTIR to confirm the absence of any crosslinked conformational state that would render the silk insoluble in water or inhibit the utility of downstream coagulation techniques. These solid films were treated with methanol to demonstrate the ability to form crosslinked water-insoluble structures. The measured concentrations of the recovered solutions are grouped by mass of initial silk powder loading and are plotted below in Figure 6. For reference, the molecular weight distributions of both the "as purified" and "autoclaved" silk systems are also included in Figure 6.

The films prepared in Fig. 6 were evaluated on a Jasco FTIR instrument to confirm the structure. As depicted in Fig. 7, the amide I and II regions exhibit characteristic peaks centered around 1650 cm ^-1 and 1525 cm ^-1 for the as-cast films, suggesting a largely amorphous "silk I" conformation (i.e., non-crosslinked). However, when these films are treated with 90% v/v methanol for 1 h, the amide I region shifts to the right and comprises a small shoulder at 1700 cm ^-1 , whereas amide II shifts to a broader shoulder at 1535 cm ^-1 . These methanol-induced changes are due to the formation of the β-sheet structure characteristic of the "silk II" conformation.

6-month foam stability. An experiment was conducted to compare the solubility of silk powders formed by milling freeze-dried silk at different boiling times and temperature conditions after 6 months of storage. Figure 8 shows the results from foams formed using primary drying only, while Figure 9 shows foams formed using primary and secondary drying.

6-month film stability. Experiments were conducted to compare the solubility of thick and thin silk films under various temperature conditions as shown in Fig. 10 after 6 months of storage.

Example 2. Blood recovery from silk-blood samples

Materials and Methods. Fresh donor blood (Vacutainer tubes, Na-heparin anticoagulant, Research Blood Components, Brighton, MA) was transferred from a single donor within 1 hour of withdrawal. The tubes were then immediately centrifuged at 1000xg for plasma isolation. An aliquot of the plasma was then immediately frozen for use as a positive control. Silk solutions were prepared from cocoons of the silk worm Bombyx mori as described above. Cocoons were purified by boiling in a solution of 0.02 M Na ₂ CO ₃ for 10, 20, 30, or 60 minutes. Silk films were cast onto polydimethylsiloxane (PDMS) surfaces, with 100 μl of blood or plasma mixed with 1.9 mL of silk solution (~8 wt % in water, regardless of boiling time), then 2 mL were cast as thick films (3 x 2.5 cm diameter casting surfaces) or thin films (3 x 7.5 cm diameter castings on surfaces). After drying the films overnight (8 h), the samples were split into 40 mg coupons for analysis. In preliminary experiments, 10 mg and 40 mg coupons were prepared from plasma and blood films for ELISA readout to tune the mass loading required for analysis within the standard curve, and the 40 mg spot was found to be adequate for recovery studies (data not shown). The films were added to 960 μl of PBS as a diluent, vortexed for 5 s, and then assayed immediately using the available supernatant. For all ELISAs (abCAM or R&D Systems in Table 1), frozen plasma aliquots were diluted according to the manufacturer's recommendations (1:100 to 1:8000), with lysed blood and plasma films tested at 40x lower dilutions based on theoretical recovery estimates.

[Table 1]

Calculation of Recovery of Plasma and Blood. In order to accurately convert the levels of protein recovered from blood and plasma coupons to the concentrations found in donor-matched plasma and thus to determine the efficiency of recovery, several physical measurements of blood and plasma were made. The wt/vol fraction (weight %) of blood or plasma (i.e., grams of blood or plasma solids per unit volume [mL]) was determined by drying a known volume of whole blood or whole plasma for 24 hours in a fume hood and measuring the residue. The weight fraction of plasma solids per blood volume was then estimated by assuming 55% plasma volume in blood. The plasma solids per weight of each film was calculated by assuming that ~20 mg of whole blood and 144 mg of silk are found in each 100 μL of blood casting solution, and the same is true for plasma casting solution (according to the values in Table 2).

[Table 2]

*

Finally, the theoretical plasma or blood load in the assay can be calculated from the fractions above. The plasma solids in each 20 mg coupon is multiplied by the plasma concentration [mg/mL] measured in the assay and then normalized to the total plasma solids mass in a given [1.02 mL] PBS resuspension volume. These calculations are presented below to illustrate the assumptions made in the analysis and to show how physical blood and plasma parameters are included to determine accurate recovery estimates from the theoretical load values.

Sample recovery was calculated as described in the section on calculating recovery of plasma and blood. Sample variance was calculated based on triplicate readings from replicate samples from the same donor. The results were compared between films prepared using different boiling times and different thicknesses (“thick” vs. “thin”) using two-way ANOVA with multiple comparisons and Tukey's correction testing at the α=0.05 family-wise significance level.

Results. For CRP and fibrinogen as model markers, we found that the recovery from silk was dependent on both boiling time and film thickness to a statistically significant degree (see Figure 11); furthermore, for CRP, the variables thickness and boiling time had a strong interaction effect on both blood and plasma recoveries, suggesting additional features in improving the recovery from the silk matrix. For specific comparisons, silk boiled for 60 minutes formed matrices that provided the highest recovery independent of boiling time and thickness, whereas lower boiling time systems decreased the recovery in a dose-dependent manner. Although not significant for all boiling times, thicker films were particularly effective in releasing the analyte reservoir compared to their thinner counterparts, regardless of sample type (blood vs. plasma).

Example 3. Stabilization of basic fibroblast growth factor (bFGF) in silk fibroin solution

Silk solutions were prepared from the cocoons of the silk worm Bombyx mori described above. The cocoons were purified by boiling in 0.02 M Na ₂ CO ₃ solution for 10, 20, or 60 minutes. The silk solutions were 1 w/v% or 4 w/v% containing 1x PBS or no buffer. The silk solutions were as purified or autoclaved. The following controls were used: 0.1% BSA in 1x PBS, 1.0% BSA in 1x PBS, and 1x PBS. bFGF was added to each solution to a final concentration of 250 ng/mL. All bFGF-containing solutions were stored at 37°C for 1 week prior to recovery measurements. Figure 12 illustrates the recovery of bFGF from silk fibroin solutions, demonstrating that lower molecular weight silk fibroin leads to higher recoveries, specifically 1% low MW silk in PBS yielded the most promising recovery of bFGF, regardless of whether the silk used was autoclaved or as-purified (i.e., “pure silk”). The red dashed lines in Figures 12a and 12b illustrate the values for the PBS control.

For the gelation monitoring experiment, bFGF was not added. Gelation was monitored using the absorbance at 550 nm (Matsumoto+, 2006). Figures 13a and 13b show the gelation results for the purified silk fibroin solution as is and the autoclaved silk fibroin solution. Figure 14 shows the gelation status for (a) the purified silk fibroin solution and (b) the autoclaved silk fibroin solution at 4°C, 22°C, and 37°C, respectively.

All patents and other publications mentioned in the detailed description and examples are incorporated herein by reference for any purpose. These publications are provided solely for their disclosure prior to the filing date of the present application. In this regard, nothing should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date of these documents or statements as to the contents thereof are based on information available to the applicant, and no corrections as to the date or contents thereof are permitted.

Example 4: Stabilization of blood and blood components in a silk fibroin matrix

Cellulose-based technology currently dominates the market share of biospecimen preservation. Dried blood spot (DBS) specimens are collected by applying a few drops of blood drawn from a finger, heel, or toe with a lancet onto specially formulated absorbent filter paper. These paper matrices may be designed to have proprietary drugs and/or built-in enzyme inhibitors. Once dried, in the laboratory, a technician uses an automated or manual hole punch to separate small discs of the saturated paper from the sheet and drops the discs onto flat-bottomed microtiter plates. The blood is eluted in buffered saline overnight at 4°C. The resulting plate containing the eluted material forms a "master" from which dilutions can be made for subsequent testing. Thus, filter paper has been used to collect blood for individual diagnostic and public health purposes since the 1960s (Guthrie and Susi, 1963). The CDC maintains an active list of substances stabilized using DBS (compiled from references over the past 20-30 years) on its website (www.cdc.gov/labstandards/nsqap_bloodspots.html), and the CDC maintains an independent quality control program for blood spots. The cards were originally manufactured and marketed by Whatman® as "Whatman FTA Card Technology" and have been repackaged and sold by GE Healthcare, GlaxoSmithKline, and Alturas Analytics. Newer companies such as Ahlstrom are also marketing fiber composites with similar liquid filtration and stabilization capabilities (www.ahlstrom.com/en/products/enduseApplication/medicalAndHealthcare/laboratoryAndDiagn osticsFiltration/Pages/Diagnosticfiltration.aspx).

For over 30 years, researchers have studied the temperature/stability relationships of markers for many disorders of DBS samples, with a particular focus on novel screening applications due to the need for low-volume analysis and remote diagnostics. To date, researchers have found that paper formats are not adequate to cover all analytes of interest, and that some markers are poorly recovered using chemically modified cards. Several specific shortcomings of these paper-based systems have also been revealed over many years of evaluation in a broad set of clinical situations. DBS specimens are limited in the achievable lower limit of detection (LLOD) and the dynamic range over which adequate quantitative discrimination can be achieved (Andreotti et al., 2010). Ensuring specimen integrity also remains a transport limitation in molecular testing for RNA viruses (Dineva et al., 2007; Puren et al., 2010). Accordingly, there is a need for improved techniques and/or products that can stabilize biological samples and/or components thereof (e.g., at ambient conditions) and allow for the recovery of sufficient quantities of a variety of analytes from the stabilized samples for detection and/or analysis.

In large-scale screening studies (e.g. epidemiology or toxicology), due to the lack of skilled local phlebotomists or general aversion to invasive sampling techniques such as venipuncture, very small capillary blood samples are obtained from most patients by finger or heel prick. In addition, human whole blood and blood components can be very labile, requiring special handling to maintain and preserve the quality of the sample for analytical evaluation of human health. The conventional method of storing and handling blood or blood fractions (plasma, serum, leukocyte buffy coat, hematocrit, etc.) is by freezing (-20°C), or in the case of very labile RNA, by freezing at ultra-low temperatures (-80°C) to preserve the integrity of the sample. Transporting biological samples from the collection site (field, hospital, etc.) to the laboratory for direct analysis or for extraction of additional plasma proteins/RNA is often very costly, as it requires the use of bulky materials such as Styrofoam boxes or dry ice. Such bulky packaging often results in expensive shipping rates (see Table 3), even for local shipments.

[Table 3]

In some developing countries or remote field settings, the minimal infrastructure to support continuous cold storage does not exist, limiting the range of diagnostic precision that can be performed. Unfortunately, it is precisely these limited resource environments that urgently need inexpensive/durable storage formats for pharmacokinetic (PK) screening or to monitor therapeutic efficacy and guide appropriate transitions to second-line therapies. Furthermore, population-wide epidemiological screening conducted by federal organizations, such as the Centers for Disease Control and Prevention (CDC), would greatly benefit from a more streamlined approach to sample stabilization that relies solely on the recovery of small, noninvasive sample volumes (Mei et al., 2001). If these organizations could be provided with a simple, inexpensive toolset to ship biological materials, even if limited, through existing infrastructure settings, we could see a paradigm shift in global health monitoring and facilitate more international phase II/III clinical studies.

In this study, the inventors disclose experiments demonstrating that properly prepared and reconstituted silk fibroin can stabilize whole blood and blood components over a range of storage temperatures and for a given time period, and can enable recovery of the components for analysis. The silk protein solution is mixed with these components in a liquid state, and then air-dried into a bio/silk complex within a thin film that functions as a stabilizing format. Alternatively, the reconstituted silk solution containing nucleic acids can be lyophilized to provide a rapid coagulation technique demonstrating that labile low-abundance nucleic acid molecules can likewise be stabilized using silk proteins. These embodiments of silk stabilization described herein further expand the platform based on recent discoveries regarding the unique properties of silk proteins as active agents and/or prophylactic agents for biological samples. In addition to using silk proteins as prophylactic agents or stabilizing agents, the inventors herein demonstrate that control of the silk processing characteristics can at least in part determine the solubility characteristics of the final stabilizing matrix, which in turn can impact the ability to recover biological samples for analysis in some therapeutic settings as well as diagnostics.

In one embodiment, the inventors have specifically demonstrated that high levels of plasma proteins can be isolated from silk-stabilized whole blood or plasma compared to a frozen liquid plasma storage format (the gold standard); for several markers evaluated, the silk-stabilized format improved retention of low-abundance plasma proteins compared to the gold standard. For nucleic acids, surprisingly, excellent recovery of RNA trapping storage was found in the presence of the RNAse-inactivated silk solution, regardless of storage temperature. Taken together, these findings indicate that reconstituted silk solutions may be formulated for a variety of products, including those in the diagnostic field, specifically in the area of care diagnostics, storage and transport of samples to other locations, and/or for caring for patients in remote areas where improved diagnostics are needed for preserved biospecimens. In some embodiments, blood samples stabilized in silk can help avoid the cold chain (i.e., continuous, uninterrupted refrigerated storage) that links remote environments where blood is drawn (via finger prick or venous blood draw) to a central laboratory where blood components can be analyzed for markers of patient health.

The only other technology currently being marketed for dry storage of blood is a paper-based technology (Whatman® FTA Dried Blood Spot Card), but this technology has not been widely adopted because it does not match the cold-chain performance metrics. In this sense, silk stabilization technology could be used to replace on-paper devices, shifting the paradigm away from relying on cold-chain storage and opening up new markets for silk devices. Additionally, some embodiments of the various aspects presented herein may be applied to other forms of sample collection, such as from urine, saliva and other bodily fluids or cells, for similar diagnostic utility.

Example 5. Silk treatment control for improved/optimal solubility of silk fibroin

The results of different processing conditions on the performance capabilities of materials derived from silk fibroin have been previously evaluated, and these performance metrics include modulation of mechanical performance, degradability, and biorelease characteristics (Altman et al., 2003; Pritchard et al., 2013; Vepari and Kaplan, 2007). However, there is no literature defining a strategy for genetically engineering a solid matrix derived from silk for the express purpose of optimizing its solubility for the purpose of trapping and recovering (any type of) bioagent in a custom and complete manner. The present inventors have found that the solubility characteristics of silk-derived matrices can be manipulated by protein-specific processing parameters, some of which have been previously described in the literature (although not used for this purpose), and other novel modifications are described herein. Herein, it is shown that the modulation of protein processing (which augments previously available techniques) provides an optimal means by which silk materials can be solubilized and trapped to yield meaningful extractable/analyzable materials for a variety of purposes. Additionally, storage experiments were conducted using representative silk film and freeze-dried foam formulations to verify stability under various temperature conditions that may be encountered when used in a field environment.

In this study, the effect of scouring time on silk solubility was evaluated. In particular, silk solutions were purified as previously described (Pritchard et al., 2013) while varying the boiling time. Briefly, cocoons of B. mori silkworm silk were purchased from Tajima Shoji Co., LTD (Sumiyoshi-cho, Naka-ku, Yokohama, Japan). The cocoons were scoured by boiling in 0.02 M Na ₂ CO ₃ solution for 10, 20, 30, or 60 minutes. The scoured fibroin was washed and then dried overnight under ambient conditions. Dried fibroin was dissolved in 9.3 M aqueous LiBr, dialyzed against distilled water for 2.5 days using Slide-A-Lyser dialysis cassettes (molecular weight cut off (MWCO) 3.5 kDa (Pierce Thermo Scientific Inc., Rockford, IL, USA), and centrifuged. Silk fibroin concentration (in w/v) was determined by evaporating water from a known volume of solution sample. All solutions were stored at 4°C before use.

Silk matrices were prepared by film casting or lyophilization methods using approximately 10 to 60 min-boiling (MB) solutions. A 60 MB solution was also autoclaved using standard 20 min autoclaving conditions as a sterile alternative. Silk films were produced by pipetting 4 wt/v % silk solutions in triplicate onto polydimethylsiloxane (PDMS) surfaces. The thin films were designed to be as spreadable as possible on the PDMS surface while still being removable as a continuous film structure. Conversely, the thick films were designed to be as thick as possible under routine pipetting procedures. These two film casting procedures represent real-world scenarios encountered when pipetting samples from multi-well plates, petri dishes, or possibly under field conditions. The casting techniques are schematically illustrated in Figure 15.

For the lyophilized foam samples, 1.5 mL of a 4 w/v% solution and a VirTis Genesis 25L Super XL freeze dryer were used, using a previously described protocol to stabilize monoclonal antibodies in silk foam (Guziewicz et al., 2011). Samples were initially frozen at -20°C and in a standard lab freezer, followed by maintaining sample cooling to -45°C using the lyophilizer before initiating primary drying (vac 100mT, -20°C). Half of the samples were complete at this point, while half were further exposed to secondary drying (vac 100mT, 35°C) to remove any residual bound water from the silk.

For both silk films and lyophilized foams, samples were removed at designated time points (0 day, 6 months, and 1 year hold) after being held at different storage temperatures (4°C, 25°C, 37°C). Silk films were cut and weighed into 40 mg samples using an analytical balance. Silk foams were ground using an analytical mill for 15 s and weighed into 15 mg samples using an analytical balance. For both, a starting mass of 60 mg per sample was assumed. All samples were mixed with 1 mL of ultrapure water (UPW), vortexed for 10 s, and centrifuged at 1000xg in 2 mL Eppendorf tubes for 10 min. To assess re-solubility, a 250 μL aliquot was removed and cast onto an additional PDMS surface for determination of silk w/v % as was done for initial silk concentration assessment.

Results and Discussion

Prior to encapsulation of biologics, attempts were made to find a silk purification technique and a method for preparing a composition that can obtain a fully-soluble silk-based material format. As shown in Figures 16 and 17, by boiling the silkworm cocoons for an increasing time (≤ 60 min) at the initiation of the purification scheme, the solubility of the film and the foam, respectively, can be effectively increased.

Also, the solubility of thin and thick silk films was compared after 6 months of storage under various temperature conditions as shown in Figs. 18 and 19. These results indicate that the solubility characteristics of silk films are very stable regardless of boiling time, and that these characteristics will not impede the ability to recover trapped biologics when high boiling times are selected for field applications.

After evaluating the behavior of the reconstituted silk fibroin material, some practical considerations regarding solubility and temperature stability are summarized below:

- Samples formed as solid matrices, such as films and foams (or powders thereof), can be used as storage-stable silk preparations for dispensing and/or on-demand use, and they are generally much more stable than silk in solution form (typically having a shelf life of 3 to 4 months before gelation). Furthermore, solid matrices are much more resilient to long-term transportation conditions, which often involve shaking in addition to fluctuating temperature profiles.

- Long-term temperature stability may be desirable in field conditions where silk materials are used as trapping matrices and recovery of analytes depends in part on sustained solubility properties.

- The solubility of silk in the presence of or in mixtures with other trapped substances may depend on, for example, the ratio of trapped biologic to silk, the type of trapped substance (e.g., but not limited to, urine, blood, feces, earwax, purified or non-purified DNA/RNA/antibodies, therapeutic agents, etc.), and/or buffers/excipients commonly applied to help stabilize these biologics.

The solubility of the silk-based material or matrix can be optimized, for example, by:

Reduce the loading of the film or foam in water (e.g., less than 4% or less than 1.5% w/v, respectively). The inventors used these values for this study, but the loading saturation can be used to optimize solubility.

Means to accelerate the drying rate of the film, such as forced air, reduced humidity (lower than ambient), increased temperature, etc. While not wishing to be bound by theory, it is thought that this mechanism is probably responsible in part for the improvement in thin film solubility demonstrated herein.

Reduction of exposure time to freeze-drying conditions and/or conditions that can induce crystallization in silk-based materials to improve foam quality.

Boiling times or methods greater than 60 minutes to selectively remove high molecular weight fractions of silk (e.g., medium chains and/or long hydrophobic sequences) during purification by enzymatic digestion, filtration, chromatography, etc.

Sterilization means, including, for example, autoclaving and sterile filtration, which may assist in continuing to reduce molecular weight and/or remove insoluble particulates.

Example 6. Stabilization of whole blood and plasma among silk fibroin-based materials

In the diagnostic application space, it is desirable to demonstrate that solubility-optimized silk matrices can also be used to stabilize human whole blood and blood components over long periods of time and under adverse environmental conditions. To this end, the inventors first conducted preliminary studies to demonstrate the scale of clinically relevant analytical tools that can be used to measure the relative abundance of plasma and blood proteins from multiple donors as soon as they are recovered from a specific format of reconstituted silk. These measurements should be consistent with measurements made on donor-matched fresh or frozen plasma, a format currently used in clinical post-processing. To demonstrate this functionality, plasma was isolated from donor venous blood and aliquots of whole blood or plasma (in an equivalent volume to a blood prick) were trapped within silk fibroin. The silk/blood or silk/plasma composite matrices were then air-dried. These matrices were then re-dissolved in a buffered saline solution and the analytes were recovered in solution.

Initially, a standard ELISA well-plate method was applied to demonstrate an immuno-affinity-based technique to identify markers in blood separately from silk proteins, or conversely, to identify specific markers that may be inhibited due to silk interference. The ELISA results were then compared to a high-throughput approach for biomarker quantification. In particular, the Luminex™ platform was used as a high-throughput method, as it applies a similar antibody-based detection, except that it has the ability to simultaneously multiplex multiple tags within the same sample. In principle, the Luminex™ approach enables the detection of more analytes and requires less sample. Ultimately, this sample multiplexing enables faster detection of diseases (e.g., but not limited to, cardiovascular disease, CVD) where multiple complementary and confirmatory biomarkers are required for diagnostic confidence. Additionally, these experiments address issues of practical importance, such as whether the presence of a stabilizing matrix (e.g., residue from a solubilized silk film) may impair the usability of the Luminex™ platform due to potential clogging of the applied fluid by high molecular weight protein residues.

For the experiments described above, fresh donor blood (Vacutainer tubes, Na-heparin anticoagulant, Research Blood Components, Brighton, MA, USA) was transferred from three separate donors within 1 hour of collection. The tubes were then immediately centrifuged at 1000xg for plasma isolation. An aliquot of the plasma was then immediately frozen to serve as a positive control. Silk solutions were prepared from cocoons of Bombyx mori silkworms as previously described (Lu et al., 2010). Silk films were cast onto a polydimethylsiloxane (PDMS) surface, where 100 μL of blood or plasma from each of the three donors was mixed with 1.9 mL of silk solution (7.6 wt % in water), and then cast over a large surface (8.5 cm diameter petri dish) for air-drying. After drying the films overnight (8 hours), samples were punched for analysis. For example, 10 mg and 40 mg coupons were prepared from plasma and blood films for ELISA readout to calibrate the mass loading required for analysis within the standard curve. For all ELISAs (R&D Systems), frozen plasma aliquots were diluted 1:10 and 1:100, while blood and plasma dissolved in silk-based films were tested undiluted.

[Table 4]

ELISA markers, protein characteristics and detection limits

High-throughput analysis (e.g., using the Luminex™ CVD kit; Table 5 below) was performed on dried film samples and plasma aliquots. The Luminex™ assay was performed according to the manufacturer's instructions (EMD Millipore Corporation, Billerica, MA, USA), which required dilutions (1:2,000 and 1:20,000) of frozen plasma aliquots to fall within the standard curve detection limits. Twenty mg blood and plasma coupons were each placed in 1 mL phosphate buffered saline (PBS), vortexed for 15 seconds, and then allowed to stand for 2 minutes for visual inspection of lysis. Similar to the plasma fractions, lysed blood and plasma in silk-based coupons (and blank silk controls) were diluted 1:200 and 1:2,000 in kit reagents to confirm potential recoveries based on previous findings in silk film format.

[Table 5]

Luminex™ Markers, Protein Characterization and Detection Limits

Calculating Recovery Rates of Plasma and Blood: As described in Example 2 above, some physical measurements of blood and plasma were first made to accurately convert the values of protein recovered from blood and plasma coupons to the concentrations found in donor-matched plasma and thereby determine the recovery efficiency.

Statistics for Luminex™ Assay Results: A two-way ANOVA was run to compare sources of sampling error between two sets of independent variables: i) the levels of blood or plasma detected across the three donors and ii) the blood or plasma coupon recovery compared to the theoretical coupon recovery as calculated from readings taken from frozen plasma aliquots. The sampling variation was calculated based on triplicate readings from the Luminex™ Assay and this error was included in the coupon recovery calculations without adjustment for variation in physical parameters such as blood and plasma weight fractions. Results were considered significant at a P value < 0.05.

These results indicated that thin films formed by boiling silk for 30 min (casted over the entire surface of a PDMS-coated 8.5 cm petri dish) would be ideal for applications where complete dissolution is desired. Films formed from silk/blood and silk/plasma mixtures were in a readily dissolvable format for further analysis (see Figure 20). Less than 40 mg of dry matrix was used in this format.

ELISA Results: The recovered liquid fractions were then tested by ELISA according to the following manufacturer's protocol for human plasma (R&D Systems, Table 4 above). As per the manufacturer's instructions, plasma samples from donor-matched blood were diluted 1:10 and 1:100 in PBS and used to determine the recovery efficiency from silk coupons.

The graph in Figure 21 shows that the ELISA kit is capable of detecting clinically relevant amounts of selected biomarkers across a range of molecular weights (16 to 150 kDa) and concentrations (5.91 ng/mL to 16.7 mg/mL). Notably, these values are consistent with the donor profile (Asian, female, 25 years old, asymptomatic) including high leptin levels and low CRP levels (Table 6), indicating the accuracy of the assay and proper preparation of the plasma. More importantly, the values of the recovered samples after blending, drying, casting, and re-solubilization of the silk were similar to the direct measurements of donor plasma. For both the approximately 10 mg and approximately 40 mg coupons (n=6 assay measurements each), values fell within the assay standard curve, and the % recoveries were calculated by averaging the means of the 10 mg and 40 mg groups and normalizing to the theoretical load values (summarized in Table 6 below). For CRP and Serpin E1, only 40 mg coupons released enough blood to fall within the detectable range of the ELISA, and therefore % recoveries are reported for a single film mass.

[Table 6]

Summary of ELISA results

Luminex Experiments: The Luminex™ System was run on six markers across three different kits and three different donor blood samples, and the results of these assays are shown in Figure 22.

Prior to trapping in the silk film, differences in the physical appearance of the blood from the three donors were observed - following venous collection and transport, donor 3 showed clear signs of hemolysis (i.e., reddening of the isolated plasma and RBC smearing on the inside of the vacutainer tube). The Luminex assay was found to be sensitive both to donor-to-donor variation and to the relative amounts of blood proteins isolated from whole blood versus proteins isolated from purified plasma across all donors. All theoretical and measured values for plasma were approximately 60 to 100% greater than the actual blood concentrations (due to removal of the cytoplasmic fraction).

Two-way analysis of variance showed significant (and in most cases very significant) variation between donors for both blood and plasma comparisons as measured by the Luminex system, except for the sVCAM marker for blood and plasma coupons and the tPAI-1 marker for blood coupons. This was due to the sensitivity of the tPAI-1, sVCAM-1 and haptoglobin assays and the standard curves applied. For example, donor 2 had plasma haptoglobin levels that exceeded the lowest assay standard [50 ng/mL] even at the 1:20,000x dilution, which made theoretical comparisons less accurate. Conversely, the detection of sVCAM-1 and tPAI-1 suffered from low sensitivity such that all plasma values at the 1:20,000x dilution were at or below the lowest assay standard (0.08 and 0.016 ng/mL, respectively); This ultimately led to difficulties in quantifying coupon recovery with high confidence (CV values > 20% for most donors and coupon types). The absence of any measurable background for silk indicates that silk proteins do not interfere with the Luminex™ instrument.

Two of the markers in the CVD panel, SAP and CRP, showed excellent agreement between theoretical load levels (based on frozen plasma aliquots) and levels recovered from blood and plasma coupons (with silk fibroin). For these two markers, two-way analysis of variance showed a significant source of error from donors, but no significant differences between frozen plasma and blood or plasma coupons (p<0.05). The differences between donor fibrinogen and haptoglobin levels were also much larger than the respective differences between matched blood and plasma coupon levels, as donor differences accounted for >60% of the variation within the sample set. These findings confirm previous results with ELISA kits, indicating adequate recovery of the analytes provided by the cast/dry/resolubilize methodology. Furthermore, the CRP assay showed excellent accuracy between coupons and frozen plasma controls, despite donor levels in the double-digit range. The above issues with the sVCAM-1, tPAI-1, and haptoglobin assays reduced our ability to detect donor-to-donor differences; therefore, in these three cases, variation from donor-to-donor differences was not considered a statistically significant contributor to overall sample variation than accuracy between plasma and frozen aliquots. Taken together, these six different CVD markers demonstrated that the Luminex platform can be used for stable blood detection in our silk system, although some donor-level correction is required.

When comparing absolute plasma levels calculated from the ELISA format to the Luminex format, lower concentrations were consistently found for all markers using the ELISA compared to the Luminex assay (see Figure 23). These differences were observed independently of location, depending on the marker used (CRP and PAI-1 are shown as examples) and the dynamic range of each assay (from 10 to 100,000 ng/mL). The differences observed between ELISA and Luminex varied in magnitude depending on the analyte (e.g., a 10-fold difference for CRP but only a 2- to 3-fold difference for PAI-1).

Surprisingly, large differences were found across all markers between the plasma isolated from donors 1 and 2 and the hemolysed plasma from donor 3, which was quite striking for CRP. The physiological role of CRP is to bind phosphocholine expressed on the surface of dead or dying cells or in their nuclear components to activate the complement system (Chang et al., 2002); therefore, it is possible that cleavage to cellular fractions (WBC or RBC) during blood collection resulted in CRP binding to the pelleted cell lysate during plasma preparation and thus was not available for detection in plasma. However, since the blood coupons also showed very low CRP levels, it is also possible that in vitro damage to the RBCs during collection or handling resulted in an overall dilution of the CRP levels for the donors, or that the donor CRP levels were simply inherently low. The ELISA screen for CRP confirmed the corresponding lower values for donor 3 as recorded by the Luminex kit, as shown in Figure 22. Additionally, the ELISA results showed that donor 3 contained higher lipid levels as evidenced by a 2-fold increase in leptin compared to donors 1 and 2 (data not shown). Without wishing to be bound by theory, lipolysis may be the cause of inaccurate CRP readings by the ELISA method (reviewed in [Martinez-Subiela and Ceron, 2005]), whereby doping of donor plasma with various lipids, such as bilirubin, resulted in a dose-dependent decrease in CRP measurements.

Time course results: After 30 days of storage at 37°C or room temperature, the Luminex™ system was run on six markers between three different donor blood samples and the results of these assays are shown in Figure 24. Similar trends as originally observed at day 0 were measured at day 30 (Figure 22). The Luminex™ assay was sensitive to both inter-donor variation and to the relative amounts of blood proteins isolated from whole blood versus proteins isolated from purified plasma across all donors (data not shown). In some embodiments, the tPAI-1 and VCAM-1 markers were detected by decreasing dilutions of these samples. Some background from the blank silk control was measured in the tPAI-1 assay, indicating some cross-interference between the antibodies used for this marker, which was exacerbated at low analyte levels. For tPAI-1, baseline silk film negative control was reduced from all film-containing samples, but coupon tPAI-1 levels were still elevated 100% compared to baseline.

All values for the analytes measured from coupons and converted to theoretical load levels showed a complete 100% recovery, with the exception of blood coupon measurements for CRP and haptoglobin. The possible biological basis for these discrepancies is discussed above. Two-way analysis of variance showed significant (and in some cases highly significant, P<0.01) differences between the different storage formats. Specifically, fibrinogen and sVCAM-1 levels measured from silk-based coupons appeared insensitive to storage conditions, whereas their respective liquid plasma levels decreased and were significantly higher at 37°C.

As shown in Figure 24, the frozen plasma aliquot levels measured at day 30, represented by the gray dashed lines on each graph, did not always match the day 0 baseline values. Specifically, the CRP levels were 80% of the day 0 baseline, whereas the fibrinogen levels were only 48% of their respective baselines. This indicates significant degradation of fibrinogen using the gold standard freeze/thaw technique, and suggests that the use of film stabilization techniques should be avoided. Indeed, previous reports have suggested that small precipitates can form in plasma isolated from heparinized blood frozen at temperatures below -80°C (e.g., the -20°C conditions used herein), and that this is generally a major contributor to loss of viability/regeneration of clotting factors (Palmer et al., 1993). This behavior is highlighted by the degradation of the corresponding fibrinogen marker taken from liquid plasma levels at room temperature and 37°C. The sensitivity of fibrinogen and other coagulation factors can be evaluated as a function of the choice of anticoagulant used and storage temperature compared to the silk system.

At 4 months (Figure 25), levels of protein markers recovered from the silk system were in many cases reduced, but still showed marked performance improvement across gold standard cold storage conditions for fibrinogen and very high levels of retention for other markers such as SAP.

Example 7. Stabilization of RNA in silk fibroin-based materials

This example demonstrates that RNA can be trapped and recovered in a lyophilized silk solution without loss of function. A series of experiments were conducted to establish optimal solution conditions to maximize recovery of RNA. For example, green fluorescent protein (GFP)-encoding mRNA was mixed with a silk solution that had undergone a 3-day mixing, lyophilization, and recovery protocol. Following this protocol, the mRNA was recovered by re-solubilization in ultrapure water, and the encapsulation and recovery efficiency was quantified using an RNA-specific fluorometric assay. In addition, the incorporation of commercially available RNAse inhibitors was applied to mitigate damage to the RNA in solution during the lyophilization protocol. Using these inhibitor/silk systems, the maintenance of RNA function following extraction from the silk was assessed by transfecting model cell lines with the recovered fraction of mRNA molecules. In some embodiments, the methods and/or compositions described herein can be stably maintained over an extended period of time (e.g., 30 to 120 days).

For the purposes of this experiment, GFP-encoding RNA was purchased from a commercial source (Stemgent® eGFP mRNA, www.stemgent.com/products/show/222). This RNA sequence was originally developed by Warren et al. to provide a simple, non-integrating (i.e., non-viral or DNA-based) strategy for reprogramming cells based on the administration of a synthetic RNA modified to overcome the innate antiviral response (Warren et al., 2010). The sequence contains three key components: i) a 5' guanine cap for improved stability in the cell cytoplasm, ii) a strong Kozak consensus sequence to initiate translation, and iii) a terminal sequence required for mRNA polyadenylation. The GFP sequence was synthetically produced with this construct using an in vitro transcription reaction templated by PCR amplicons. The concentrate is sold in high concentrations (100 jig/mL) by Stemgent®, and previous results by Warren et al. suggest that translation of sufficient GFP can be achieved within a short period of time (8 to 12 h), making it ideal for encapsulation and retrieval studies requiring assays for RNA-specific function.

Silk solutions were prepared using boiling and dissolution conditions (9.3 M LiBr, dialyzed against diH ₂ O for 48 hours) for 20 minutes and the final concentration was 6 wt % in ultrapure water, which was assessed by drying a measured volume of solution. This solution was diluted to 2%, 1%, or 0.5% wt/v using guaranteed RNAse-free water. Tris-EDTA (TE) was added to achieve final working concentrations of 10 mM and 1 mM, respectively. In some embodiments where the silk purification process cannot be used with a DEPC-based RNAse treatment, for example, due to the required autoclaving step which can alter the silk protein, other methods may be used to eliminate the presence or activity of RNAse during mRNA handling. For example, guaranteed RNAse/DNAse-free pipette tips, tubing, and solutions were used as purchased from Ambion® and RNAzap was used on all external surfaces. GFP mRNA stock (supplied as 1xTE) of 60 jig was stored at -80°C until use, at which point it was thawed on ice and subsequently mixed v/v in each vessel with a 20 jI mRNA/80 jI (Silk + TE) solution for a final mRNA concentration of 20 x 10 ³ ng/mL as a final step. The mRNA pipetting process lasted less than 5 min for all samples. Matched controls without mRNA loading were also prepared using blank 1xTE buffer. In addition, these conditions were repeated extensively (silk alone and silk/RNA) with the addition of SUPERaseㆍInTM RNase inhibitor (Ambion®) to further protect the RNA during handling.

Lyophilization of Silk/RNA Solutions: Following the mixing step, all 100 μL samples stored in 2 mL tubes were immediately transferred to a Vertis Genesis 25 L Super XL freeze dryer using a previously described protocol to stabilize monoclonal antibodies in silk foam (Guziewicz et al., 2011). The samples were left with the caps open on their sides to facilitate rapid drying, and the tubes were placed in direct contact with a cooling plate to facilitate more precise and uniform heat control. The process was initiated by ramping from a -20 °C frozen condition, where the samples were slowly cooled to -45 °C over 63 minutes on the plate and held at that temperature for an additional 8 hours before being rewarmed to -20 °C. Pre-drying at -20 °C for 40 hours under 100 mT vacuum was then performed to remove unbound water. The samples were rewarmed to 35 °C and held for 620 minutes as a secondary drying step to remove bound water. Samples were then stored at 800 mT pressure at -4°C for typically 2 to 3 hours until the recovery protocol was initiated.

The concentration of the silk solution (6-1% silk w/v) was optimized to allow re-solubilization of the silk fibroin-based material after the freeze-drying protocol. As shown in Figure 26 below, independent foams were generated from each solution in the presence or absence of EDTA (in TE buffer). Upon re-solubilization in water, a small fraction of undissolved silk residue was found to be sized depending on the initial silk loading. However, the size/solubility was independent of TE buffer. Based on these findings, 2% w/v was chosen as a starting point to investigate the effect of concentration on solubility.

Recovery and Measurement of RNA Content: Following screening studies, 2%, 1%, and 0.5% silk solutions were selected as viable candidates for RNA recovery studies. Following the trapping and lyophilization protocol described above, samples were removed from the -45°C lyophilizer storage conditions and immediately thawed on ice for 2 hours (duration of assay preparation).

RNA Recovery Quantification: Quantitation-iTTM Ribogreen® RNA reagent and kit were prepared according to the manufacturer's guidelines from the web (probes.invitrogen.com/media/pis/mp11490.pdf). A standard curve (range 1000 to 7.8125 ng/mL) was generated using ribosomal RNA standards, and simultaneously, crude GFP mRNA stored at -80°C was thawed on ice, diluted 1:80, and used as an internal control to correct the results of the standard curve when changing the order of the Ribogreen reagent. Assays were performed in a 96-well format requiring 25 μL per sample. After preparing the standards, the lyophilized silk samples (and 0% control) were removed from ice, 200 μL of DNAase/RNAse free water was added, vortexed for 15 seconds, and 25 μL of sample was immediately pipetted into each well (replicate assay reads from N=3 biological replicates).

Transfection of Eukaryotic Cells Using Recovered mRNA: To demonstrate the stability of the recovered mRNA and its potential for subsequent use as an in vitro translation template, approximately 10 μL per sample was retained and immediately re-solubilized with silk/inhibitor complexes. Prior to recovery of mRNA, 293 fibroblasts were seeded at a density of ^5x104 cells/ ^cm2 in 96-well plates and allowed to attach in the presence of 15% serum, 1% antibiotic/antimycotic in Dulbecco's modified Eagle's medium (DMEM, Invitrogen). Cells were maintained at 37°C with 5% _CO2 .

The Stemfect™ Transfection Kit was used according to the manufacturer's recommendations and published user protocol (Stemgent, www.stemgent.com/products/show/221). The transfection reagent was prepared as a stock solution by combining transfection buffer and dispensed into 7.33 μL aliquots, which were then combined with 10 μL of recovered mRNA. The buffer/reagent/mRNA solution was allowed to equilibrate at room temperature for 15 minutes after which 15 μL from each aliquot was added dropwise to individual wells of a 96-well plate. The cells were then returned to incubation as above for 12 hours, at which time they were imaged on an Axiovert CFL fluorescence microscope with a filter set for 488 nm (blue) excitation and 510 nm (green) emission.

Results and Discussion

The results of the initial study are shown in Figure 27. The Ribogreen assay showed a small but significant background in most of the reads from the RNA recovered from all groups, as well as the apparent silk protein (quantitatively fluorescein at 480 nm) in the silk-only group. Therefore, the unloaded group was used to normalize to the silk-RNA samples from all respective concentrations. The RNA recovered from the 1% and 0.5% groups was comparable to the non-silk 0% group, but all of these lyophilized systems were well below the frozen stock.

Based on these results, it was attempted to increase the efficiency of RNA recovery, for example, by adjusting the solution storage conditions. It was suspected that RNAse was present in small amounts in the silk solutions and that the mRNA recovered in all conditions was prone to post-recovery degradation during assay preparation, therefore, SUPERaseㆍInTM RNase inhibitor (tools.invitrogen.com/content/sfs/manuals/sp_2694.pdf) was included in the assay at a working concentration of 1 U/μL and the results are shown in Fig. 28. Compared to previous assay runs using silk/TE alone (Fig. 27), the presence of the inhibitor significantly improved the recovery of mRNA compared to the -80°C storage conditions. Additionally, interference from the silk component was minimized, such that the values for the 1% and 0.5% groups corresponded to the non-silk 0% group.

As shown in Figure 29 below, cells in all medium conditions containing transfection reagent and mRNA, including the frozen stock (-80°C storage) and all recovered silk groups, induced GFP expression by 293 fibroblasts by 12 h of incubation. Furthermore, the -80°C group was found to produce a more extensive expression pattern in cell colonies in the seeded wells, possibly due to the dose-dependence of mRNA-based transfection and the limited dilution of the -80°C stock. The mRNA recovered from the silk group (0-2%) appeared to be the minimum titer required to observe positive transfection results.

In this example, recovery of RNA from the lyophilized silk solution was sufficient for transfection, but at absolute levels below the frozen control. However, the preparation of silk and/or the method of silk treatment can be optimized to increase RNA recovery from lyophilized silk solutions. For example, the pre-solubilization boiling time can be varied to select for the most soluble solution subtype. Figure 17 shows that boiling for 60 minutes or longer can be used to recover RNA with improved resolubilization from lyophilized silk solutions.

Specific References

All patents and other publications identified in this specification and examples are expressly incorporated herein by reference for all purposes. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. Any statements as to the date or reference to the contents of these documents are based on information available to applicants and do not constitute any admission as to the accuracy of the date or contents of these documents.

Equivalent

While the preferred embodiments have been particularly shown and described herein, it will be apparent to those skilled in the art that various modifications, additions, substitutions, etc. may be made therein without departing from the spirit of the invention and that these are therefore considered to be within the scope of the invention as defined in the following claims. Furthermore, it will be appreciated by those skilled in the art that any one of the various embodiments described and illustrated herein may be further modified to introduce features shown in any of the other embodiments disclosed herein, to the extent not already indicated.

SEQUENCE LISTING <110> TRUSTEES OF TUFTS COLLEGE <120> LOW MOLECULAR WEIGHT SILK COMPOSITIONS AND STABILIZING SILK COMPOSITIONS <130> <140> <141> <150> PCT/US2014/029636 <151> 2014-03-14 <150> 61/909,687 <151> 2013-11-27 <150> 61/883,732 <151> 2013-09-27 <150> 61/830,950 <151> 2013-06-04 <150> 61/792,161 <151> 2013-03-15 <160> 24 <170> PatentIn version 3.5 <210> 1 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (1)..(90) <223> This sequence may encompass 5-15 'Gly Ala Gly Ala Gly Ser' repeating units, wherein some positions may be absent <220> <223> See specification as filed for detailed description of substitutions and preferred embodiments <400> 1 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala 20 25 30 Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 35 40 45 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 50 55 60 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala 65 70 75 80 Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 85 90 <210> 2 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (2)..(2) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (4)..(4) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (6)..(6) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (8)..(8) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (10)..(10) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (12)..(12) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (14)..(14) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (16)..(16) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (18)..(18) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (20)..(20) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (22)..(22) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (24)..(24) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (26)..(26) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (28)..(28) <223> Val, Ile or Ala <220> <221> MOD_RES <222> (30)..(30) <223> Val, Ile or Ala <220> <221> MISC_FEATURE <222> (1)..(30) <223> This sequence may encompass 5-15 'Gly Xaa' repeating units, wherein some positions may be absent <220> <223> See specification as filed for detailed description of substitutions and preferred embodiments <400> 2 Gly 30 <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 3 Gly Ala Ala Ser 1 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> May or may not be present <220> <221> MOD_RES <222> (14)..(15) <223> May or may not be present <400> 4 Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 1 5 10 15 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(5) <223> Any amino acid and this region may encompass 1-4 residues, wherein some positions may be absent <220> <221> MOD_RES <222> (8)..(8) <223> Any amino acid <400> 5 Gly Xaa <221> MOD_RES <222> (4)..(4) <223> Ala, Ser, Tyr, Arg, Asp, Val or Trp <400> 6 Gly Gly Gly <222> (2)..(2) <223> May or may not be present <220> <221> MOD_RES <222> (3)..(6) <223> May or may not be present <220> <221> MOD_RES <222> (8)..(8) <223> May or may not be present <220> <221> MOD_RES <222> (10)..(12) <223> May or may not be present <220> <221> MISC_FEATURE <222> (1)..(12) <223> This sequence may encompass 1-2 '(Ser)1-2 (Ala)1-4' repeating units, wherein some positions may be absent <220> <223> See specification as filed for detailed description of substitutions and preferred embodiments <400> 7 Ser Ser Ala Ala Ala Ala Ser Ser Ala Ala Ala Ala 1 5 10 <210> 8 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 8 Gly Leu Gly Gly Leu Gly 1 5 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> Leu, Ile, Val or Pro <220> <221> MOD_RES <222> (5)..(5) <223> Leu, Ile, Val or Pro <400> 9 Gly Xaa Gly Gly Xaa Gly 1 5 <210> 10 <211> 300 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (3)..(14) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (5)..(5) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (8)..(8) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (11)..(11) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (14)..(14) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (18)..(29) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (20)..(20) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (23)..(23) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (26)..(26) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (29)..(29) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (33)..(44) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (35)..(35) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (38)..(38) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (41)..(41) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (44)..(44) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (48)..(59) <223> This region may encompass 1-4 'Gly Gly <222> (53)..(53) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (56)..(56) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (59)..(59) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (63)..(74) <223> This region may encompass 1-4 'Gly Gly Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (71)..(71) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (74)..(74) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (78)..(89) <223> This region may encompass 1-4 'Gly Gly MOD_RES <222> (86)..(86) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (89)..(89) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (93)..(104) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (95)..(95) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (98)..(98) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (101)..(101) <223> Tyr; Val, Ser or Ala <220> <221> MOD_RES <222> (104)..(104) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (108)..(119) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (110)..(110) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (113)..(113) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (116)..(116) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (119)..(119) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (123)..(134) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (125)..(125) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (128)..(128) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (131)..(131) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (134)..(134) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (138)..(149) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (140)..(140) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (143)..(143) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (146)..(146) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (149)..(149) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (153)..(164) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (155)..(155) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (158)..(158) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (161)..(161) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (164)..(164) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (168)..(179) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (170)..(170) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (173)..(173) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (176)..(176) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (179)..(179) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (183)..(194) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (185)..(185) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (188)..(188) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (191)..(191) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (194)..(194) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (198)..(209) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (200)..(200) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (203)..(203) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (206)..(206) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (209)..(209) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (213)..(224) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (215)..(215) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (218)..(218) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (221)..(221) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (224)..(224) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (228)..(239) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (230)..(230) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (233)..(233) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (236)..(236) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (239)..(239) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (243)..(254) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (245)..(245) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (248)..(248) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (251)..(251) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (254)..(254) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (258)..(269) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (260)..(260) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (263)..(263) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (266)..(266) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (269)..(269) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (273)..(284) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (275)..(275) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (278)..(278) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (281)..(281) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (284)..(284) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (288)..(299) <223> This region may encompass 1-4 'Gly Gly Xaa' repeating units, wherein some positions may be absent <220> <221> MOD_RES <222> (290)..(290) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (293)..(293) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (296)..(296) <223> Tyr, Val, Ser or Ala <220> <221> MOD_RES <222> (299)..(299) <223> Tyr, Val, Ser or Ala <220> <221> MISC_FEATURE <222> (1)..(300) <223> This sequence may encompass 3-20 'Gly Pro (Gly Gly Xaa)1-4 Tyr)' repeating units, wherein some positions may be absent <220> <223> See specification as filed for detailed description of substitutions and preferred embodiments <400> 10 Gly Pro Gly Gly Gly Pro Gly 35 40 45 Gly Xaa Gly Gly Xaa Gly Gly Gly Gly Xaa Gly 85 90 95 Gly Xaa Gly Gly Xaa Tyr Gly Pro Gly Gly 165 170 175 Gly Gly Gly Gly Pro Gly Gly 290 295 300 <210> 11 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (8)..(24) <223> May or may not be present <400> 11 Gly Arg Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 1 5 10 15 Ala Ala Ala Ala Ala Ala Ala Ala 20 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (10)..(10) <223> May or may not be present <400> 12 Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala 1 5 10 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (3)..(3) <223> Gln, Tyr, Leu, Ala, Ser or Arg <220> <221> MOD_RES <222> (5)..(5) <223> Gln, Tyr, Leu, Ala, Ser or Arg <220> <221> MOD_RES <222> (7)..(8) <223> Gln, Tyr, Leu, Ala, Ser or Arg <400> 13 Gly Gly Gly 1 5 10 15 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 15 Tyr Glu Tyr Ala Trp Ser Ser Glu 1 5 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 16 Ser Asp Phe Gly Thr Gly Ser 1 5 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 17 Arg Arg Ala Gly Tyr Asp Arg 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 18 Glu Val Ile Val Ile Asp Asp Arg 1 5 <210> 19 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 19 Thr Thr Ile Ile Glu Asp Leu Asp Ile Thr Ile Asp Gly Ala Asp Gly 1 5 10 15 Pro Ile <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 20 Thr Ile Ser Glu Glu Leu Thr Ile 1 5 <210> 21 <400> 21 000 <210> 22 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (4)..(5) <223> Any amino acid <400> 22 Gly Pro Gly Xaa Gly Pro MISC_FEATURE <222> (3)..(22) <223> This region may encompass 3-20 residues, wherein some positions may be absent <400> 24 Gly Gly

Claims (31)

A composition comprising a population of silk fibroin fragments having a molecular weight within a predetermined range,
Less than 15% of the total number of silk fibroin fragments in a population have a molecular weight exceeding 200 kDa;
A composition characterized in that at least 50% of the total number of silk fibroin fragments in the population have a molecular weight within a specified range of 3.5 kDa to 120 kDa. In the first paragraph,
(a) the above-mentioned range is (i) 5 to 120 kDa; (ii) 10 to 120 kDa; (iii) 15 to 120 kDa; (iv) 20 to 120 kDa; (v) 20 to 110 kDa; (vi) 20 to 100 kDa; (vii) 20 to 90 kDa; (viii) 20 to 80 kDa; (ix) 30 to 120 kDa; (x) 30 to 100 kDa; (xi) 30 to 90 kDa; (xii) 30 to 80 kDa; (xiii) 40 to 100 kDa; and (xiv) 40 to 90 kDa; or
(b) not more than 35% of the total silk fibroin fragment population has a molecular weight within the range of 120 kDa to 200 kDa; or
(c) a composition wherein at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the total number of silk fibroin fragments in the population have a molecular weight within the range specified above. A composition according to claim 1 or 2, further comprising at least one active agent, wherein the ratio of silk fibroin fragments to the active agent is from 1:1 to 1:10000. A composition according to claim 1 or 2, further comprising an additive. In claim 4, the additive is comprised at least 5 wt% of the composition, or the composition is selected from the group comprising a wound healing agent, a biological sample, an enzyme, an immunogen, a cell adhesion mediator, a stabilizer, a biocompatible polymer, a growth factor, an anti-inflammatory agent, an antimicrobial agent, a vaccine, an agent that stimulates tissue formation or repair and regrowth of natural tissue, a therapeutic agent, or a combination thereof. A composition according to claim 1 or 2, wherein the specified range has a lower limit of 20 KDa and the specified range has an upper limit of 50 kDa. In paragraph 1 or 2,
(a) the composition is a solution; or
(b) a composition characterized in that the composition is in solid form, (i) the composition has a water solubility greater than that of the reference silk fibroin composition; or (ii) the composition has a dissolution rate of 1 mg/s to 100 mg/s. An aqueous solution of silk fibroin comprising the composition of claim 1 or claim 2,
(a) silk fibroin is present in the solution at a concentration of 0.1% wt/v to 50% wt/v; or
(b) An aqueous silk fibroin solution, characterized in that the aqueous silk fibroin solution remains stable for at least 3 days, at least 7 days, or at least 2 weeks. A silk fibroin article comprising the composition of claim 1 or 2, wherein the silk fibroin article is in a form selected from the group consisting of a film, a sheet, a gel or hydrogel, a mesh, a mat, a non-woven mat, a fabric, a scaffold, a tube, a block, a fiber, a particle, a powder, a three-dimensional structure, an implant, a foam, a needle, a lyophilized article, and any combination thereof, and characterized by the ability to be resolubilized in water to form a silk fibroin solution free of silk fibroin aggregates. A composition comprising silk fibroin particles, wherein the silk fibroin particles comprise the composition of claim 1 or 2, and are characterized in that they are storage-stable for at least 1 month. A composition comprising the composition of claim 1 or claim 2 and an active agent distributed in the composition,
(a) the composition is in a form selected from the group consisting of a solution, a film, a sheet, a gel or hydrogel, a mesh, a mat, a non-woven mat, a fabric, a scaffold, a tube, a block, a fiber, a particle, a powder, a three-dimensional structure, an implant, a foam, a needle, a lyophilized article, and any combination thereof;
(b) the composition is a solution;
(c) a composition in solid form, characterized by the ability to be re-dissolved in water to form a silk fibroin solution free of silk fibroin aggregates. An article comprising a solid porous substrate, and the composition of claim 1 or 2 dispersed within the substrate, deposited on the substrate, or both. A method for forming a silk fibroin solution, comprising dissolving a silk fibroin article characterized by its ability to be re-dissolved in water of claim 9 to form a silk fibroin solution free of silk fibroin aggregates. A method for forming a silk fibroin solution, comprising dissolving a composition in a solid form, characterized by the ability to be re-dissolved in water of claim 11, to form a silk fibroin solution free of silk fibroin aggregates, in water at a non-saturated concentration, thereby forming a silk fibroin solution free of silk fibroin aggregates.
(a) the dissolved silk fibroin in the solution remains stable; or
(b) a method further comprising the step of maintaining the silk fibroin solution at room temperature or higher for a predetermined period of time. A method of stabilizing an agent for a predetermined period of time, comprising contacting the agent with the composition of claim 1 or claim 2 to form an agent-containing composition,
(a) the composition is solvent-free, or the contacting step is performed under solvent-free conditions, and the method may further comprise forming a silk fibroin solution or article comprising the composition and the agent distributed therein; or
(b) the method further comprises the step of maintaining the agent-containing composition at room temperature or higher for a period of time, or 24 hours or longer, or 1 week or longer; or
(c) the method further comprises forming a silk fibroin article comprising the composition and the agent dispersed therein, or the method further comprises maintaining the agent in the silk fibroin solution or the silk fibroin article at room temperature or higher for a period of time. Providing a solid silk fibroin composition comprising the composition of claim 1 or 2 and at least one active agent distributed in the composition;
Comprising dissolving the composition in water to form a sample solution comprising a population of silk fibroin fragments and a detectable amount of at least one active agent;
A method of recovering at least one agent. A composition comprising a group of silk fibroin fragments,
Less than 15% of the total number of silk fibroin fragments in a population have a molecular weight exceeding 200 kDa;
At least 50% of the total number of silk fibroin fragments in the population have a molecular weight within the range of 3.5 kDa to 120 kDa,
A composition characterized in that the composition is lyophilized and the silk fibroin fragments of the lyophilized composition can be dissolved in an aqueous solution. A composition according to claim 17, further comprising at least one active agent. A composition in claim 18, wherein the ratio of silk fibroin fragments to the active agent is from 1:1 to 1:10000. A composition according to claim 17, further comprising an additive, wherein the additive is comprised at least 5 wt% of the composition. A composition comprising 0.1% wt/v to 90% wt/v silk fibroin in claim 17. A composition in claim 17, wherein at least 50 wt % of the silk fibroin fragments have a molecular weight within a specified range, wherein the specified range has a lower limit of 20 kDa and the specified range has an upper limit of 50 kDa. A composition in claim 18 which is storage-stable for at least one month. A composition in claim 17 which can be re-dissolved in an aqueous solution at room temperature. In claim 17, the composition further comprises an additive, wherein the additive is selected from the group consisting of a wound healing agent, a biological sample, an enzyme, an immunogen, a cell adhesion mediator, a stabilizer, a biocompatible polymer, a growth factor, an anti-inflammatory agent, an antimicrobial agent, a vaccine, an agent that stimulates tissue formation or repair and regrowth of natural tissue, a therapeutic agent, or a combination thereof. In claim 25, the additive is a wound healing agent, wherein the wound healing agent is dexpanthenol; growth factor; enzyme; hormone; povidone-iodide; fatty acid; anti-inflammatory agent; antibiotic; antimicrobial agent; antiseptic; cytokine; thrombin; analgesic; opioid; aminooxyl; furoxin; nitrosothiols; nitrates and anthocyanins; nucleosides and adenosine; nucleotides, adenosine diphosphate (ADP) and adenosine triphosphate (ATP); neurotransmitters/neuromodulators, acetylcholine and 5-hydroxytryptamine (serotonin/5-HT); histamine, catecholamines, adrenaline and noradrenaline; lipid molecules, sphingosine-1-phosphate and lysophosphatidic acid; amino acids, arginine and lysine; A composition selected from the group consisting of peptides, bradykinin, substance P and calcium gene-related peptide (CGRP); nitric oxide; and any combination thereof. A composition in accordance with claim 25, wherein the additive is a biological sample, and the biological sample is or comprises a cell, tissue, blood, a breast milk product, amniotic fluid, sputum, urine, saliva, mucus, semen, cerebrospinal fluid, bronchial aspirate, sweat, nasal discharge, vaginal fluid, liquefied feces, synovial fluid, lymph, tears, tracheal aspirate, or fractions thereof, or combinations thereof. A composition in claim 17, wherein the composition is in a form selected from the group consisting of a film, a sheet, a gel or hydrogel, a mesh, a mat, a non-woven mat, a fabric, a scaffold, a tube, a block, a fiber, a particle, a powder, a three-dimensional structure, an implant, a foam, a needle, a lyophilized article, and any combination thereof. A composition according to claim 17, further comprising a material selected from poly(methyl methacrylate) microspheres, hydroxylapatite, poly(L-lactic acid), lipoaspirate, lidocaine, collagen, elastin, glycosaminoglycans, hyaluronic acid, botulinum toxin, and abobotulinumtoxinA. A composition in claim 25, wherein the additive is a therapeutic agent, and the therapeutic agent is an anti-inflammatory agent or an analgesic agent. A composition in claim 25, wherein the additive is a therapeutic agent, and the therapeutic agent is selected from small organic or inorganic molecules; saccharin; oligosaccharides; polysaccharides; biological macromolecules; peptide mimetics; antibodies and antigen-binding fragments thereof; nucleic acids; nucleic acid analogues and derivatives; extracts prepared from biological materials; animal tissues; naturally occurring or synthetic compositions; and any combination thereof.

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